Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted in accordance with GLP and Guidelines(OECD,USEPA OPPTS).and sufficient data is available for the interpretation of study results.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: USEPA OPPTS 870.3650
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The purity of three drums of test material used was 99.45, 99.65 and 99.65%, as determined by gas chromatography

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
CD rats (Crl:CD(SD)IGS BR) were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier (Charles River Laboratories Inc., Portage, Michigan).

Animals were about 8 weeks of age at study start. Each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a lab veterinarian, to determine their general health status and acceptability for study purposes upon arrival at the laboratory. The animals were housed two of same sex per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week prior to the start of the study.

After assignment to study, animals were housed one per cage in stainless steel cage in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). A 12-hour light/dark photocycle was maintained for all animal room(s) with lights on at 6:00 a.m. and off at 6:00 p.m. Room air was exchanged approximately 12-15 times/hour.

Cages had wire-mesh floors and were suspended above catch pans. Cages contained feed containers and pressure activated, nipple-type watering systems. Dams were housed one per cage (with their litter) in plastic cages provided with corn cob nesting material from approximately day 19 of gestation and throughout the lactation phase of the study. Room temperature and relative humidity were recorded daily. The relative humidity was maintained within a range of 40-70%, with the exception of some minor deviations that occurred during the study (data contained in study file). These deviations involved transient excursions in the range of 38-39% humidity. These deviations were minor, had no noticeable impact on the animals, and did not affect the integrity of this study. The room temperature was maintained within the range of 22 ± 1°C (with a maximum permissible excursion range of ± 3°C). These values were within the laboratory recommended range for rats.

For the exposures, animals were transferred each day to stainless steel cages placed inside Rochester-style exposure chambers (see Animal Exposure section) without access to feed or water. All animals were housed one per cage during exposures except during the mating period (see breeding procedures).

Prior to test material administration, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) which were correlated to unique alphanumeric identification numbers. If a transponder stopped functioning or was lost, it was replaced with a new transponder which was correlated with the unique animal number.

Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were provided ad libitum except during the 6 hour exposure period when feed and water were withheld. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. The results of these analyses indicated no contaminants at levels that would interfere with the conduct of this study or interpretation of the results.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Animals were exposed to the test material daily via inhalation. Each day during the exposure period, rats were transferred to whole-body exposure chambers, exposed to the targeted concentrations of test material for six hours, then returned to their home cages.

The animals were exposed to filtered air or test material vapors in 2 cubic meter stainless steel and glass Rochester-type whole-body exposure chambers [1.3 m x 1.2 m wide x 1.2 m deep with a pyramidal top and bottom]. Chamber airflow was maintained at approximately 450 liters per minute. This flow rate was sufficient to provide the normal concentration of oxygen to the animals and 12-15 calculated air changes per hour. The chambers were operated at a slightly negative pressure, relative to the surrounding area. Chamber airflow data were collected using Setra Differential Pressure Transducers (Setra System, Inc., Acton, Massachusetts). The signal from the pressure transducer was sent to the CAMILE TG4 Acquisition and Control System and recorded in liters per minute. The differential pressure transducer was calibrated with a gas meter (Singer Aluminum Diaphragm Meter, Model AL-2300, American Meter Division, Philadelphia, Pennsylvania) prior to the start of the study. Chamber temperature and relative humidity data were collected using a resistance temperature device (RTD) (Omega HX94C, Omega Engineering Inc., Stamford, CT) coupled to the CAMILE TG4 Data Acquisition and Control System. The chamber temperature and relative humidity was controlled by a system designed to maintain values of approximately 22 ± 3°C and 30 to 70%, respectively. Chamber temperature, relative humidity, and airflow data were automatically logged into data files once per hour by CAMILE, and subsequently extracted and printed for inclusion in the study file with the exception of chamber temperature and relative humidity data generated beginning July 17 through the last day of exposure. The Omega RTDs normally used to measure chamber temperature and relative humidity were inadvertently sprayed with water during post-exposure sanitization of the chambers on July 16, causing them to lose their ability to measure data. Therefore, chamber temperature and relative humidity data for each exposure chamber were manually recorded once per hour from calibrated thermometers and hygrometers stationed inside the chambers. This equipment was in place for data collection in the event of a failure of the CAMILE® TG4 Data Acquisition and Control System or its associated equipment.

The various concentrations of test material were generated using a glass J-tube method. Liquid test material was pumped into the glass J-tube assembly and vaporized by compressed nitrogen gas passing through the bead bed of the glass J-tube approximately 40 liters per minute. The nitrogen was heated as needed with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. The generation system was electrically grounded and the J-tubes were changed as needed. The compressed nitrogen and test material vapors were mixed and diluted with supply air to achieve a total flow of 450 liters per minute at the desired test chamber concentration.

Animals in the negative control group were exposed to an atmosphere of approximately 410 L/minute of humidified, HEPA-filtered air and approximately 40 L/minute of compressed nitrogen.

The chamber concentrations of test substance, measured approximately in the center of the breathing zone of the animals, was determined at least once per hour with a Miran 1A infrared (IR) spectrophotometer (Foxboro/Wilks, South Norwalk, Connecticut). The IR spectrophotometer was calibrated and a standard curve was compiled prior to the start of the study, using air standards prepared by vaporizing measured volumes of test substance into Tedlar sample bags (Series 233, SKC, Eighty Four, Pennsylvania) along with the metered volumes of dry, compressed air. The analytical concentration during the exposure was interpolated by the CAMILE TG4 Data Acquisition and Control System using the standard curve. The analytical system was checked prior to each exposure with a standard gasbag containing a known concentration of test substance. The nominal concentration of the test material in each chamber was estimated based on the amount of test material used and the total airflow through the chamber. Prior to the start of the study, each of the chambers was checked to ensure that a uniform distribution of vapor was present throughout the breathing zone of the animals.
Details on mating procedure:
Breeding of the adults commenced after approximately two weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations of test substance, measured approximately in the center of the breathing zone of the animals, was determined at least once per hour with a Miran 1A infrared (IR) spectrophotometer (Foxboro/Wilks, South Norwalk, Connecticut). The IR spectrophotometer was calibrated and a standard curve was compiled prior to the start of the study, using air standards prepared by vaporizing measured volumes of test substance into Tedlar sample bags (Series 233, SKC, Eighty Four, Pennsylvania) along with the metered volumes of dry, compressed air. The analytical concentration during the exposure was interpolated by the CAMILE TG4 Data Acquisition and Control System using the standard curve. The analytical system was checked prior to each exposure with a standard gasbag of known concentration of test substance. The nominal concentration of the test substance in each chamber was estimated based on the amount of test substance used and the total airflow through the chamber. Prior to the start of the study, each of the chambers was checked to ensure that a uniform distribution of vapor was present throughout the breathing zone of the animals.
Duration of treatment / exposure:
Males were exposed 6 hours per day for 14 days prior to mating and continuing throughout mating until necropsy for a total of 33 consecutive days. Females were exposed 6 hours per day for 14 days premating, continuing throughout mating (two weeks) and gestation (up to and including GD 20).
Frequency of treatment:
Daily, 6h/day, 7day/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50, 200, 600 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
50.1 ± 1.0, 201.0 ± 2.1, 601.8 ± 9.1 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Section: 7.5.3
Endpoint study record: Key Study_Dow_2005_Combined Repeated exposure_HET-DR-0306-9270-009
UUID: IUC5-c60d2a48-df7a-4a52-8a81-183e3264fada
Positive control:
None

Examinations

Parental animals: Observations and examinations:
See Section: 7.5.3
Endpoint study record: Key Study_Dow_2005_Combined Repeated exposure_HET-DR-0306-9270-009
UUID: IUC5-c60d2a48-df7a-4a52-8a81-183e3264fada
Oestrous cyclicity (parental animals):
Not evaluated
Sperm parameters (parental animals):
Not evaluated
Litter observations:
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. Litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, the number of live and dead pups on LD 0, 1, and 4, and the sex and body weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period. Any pups found dead or sacrificed in moribund condition were sexed and examined grossly, if possible, for external and visceral defects and discarded.
Postmortem examinations (parental animals):
See Section: 7.5.3
Endpoint study record: Key Study_Dow_2005_Combined Repeated exposure_HET-DR-0306-9270-009
UUID: IUC5-c60d2a48-df7a-4a52-8a81-183e3264fada
Postmortem examinations (offspring):
All pups surviving to LD 4 were euthanized by oral administration of sodium pentobarbital solution, examined for gross external alterations, and then discarded.
Statistics:
See Section: 7.5.3 for adult observations
Endpoint study record: Key Study_Dow_2005_Combined Repeated exposure_HET-DR-0306-9270-009
UUID: IUC5-c60d2a48-df7a-4a52-8a81-183e3264fada

Gestation length, average time to mating, and litter size were analyzed using a nonparametric ANOVA. If the ANOVA was significant at α = 0.05, the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. The mating, conception, fertility and gestation indices were analyzed by the Fisher exact probability test with Bonferroni's correction at experiment-wise α = 0.05. Evaluation of the neonatal sex ratio on postnatal day 1 was performed by the binomial distribution test (α = 0.05). Gender was determined for pups found dead on postnatal day 0 and these data were included in sex ratio calculations. Post-implantation loss, pup survival indices, and other incidence data among neonates were analyzed using the litter as the experimental unit, by the censored Wilcoxon test as modified with Bonferroni’s correction. Both the Dunnett’s test and Bonferroni’s correction correct for multiple comparisons to the control to keep the experiment-wise error rate at 0.05. Both were reported at the experimentwise alpha level.
Reproductive indices:
Female mating index = (No. females with evidence of mating/No. paired) x 100
Male mating index = (No. males with evidence of mating /No. paired) x 100
Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
Male conception index = (No. males siring a litter/No. mated) x 100
Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
Male fertility index = (No. males siring a litter/No. paired) x 100
Gestation index = (No. females delivering a viable litter/No. females delivering a litter) x 100
Offspring viability indices:
Gestation survival index = Percentage of delivered pups alive at birth
Day 1 or 4 Pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100
Post-implantation loss = (No. implantation sites – No. live born pups) x 100 / No. implantation sites

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See Section: 7.5.3 Endpoint study record: Key Study_Dow_2005_Combined Repeated exposure_HET-DR-0306-9270-009 UUID: IUC5-c60d2a48-df7a-4a52-8a81-183e3264fada
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Section: 7.5.3 Endpoint study record: Key Study_Dow_2005_Combined Repeated exposure_HET-DR-0306-9270-009 UUID: IUC5-c60d2a48-df7a-4a52-8a81-183e3264fada
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See Section: 7.5.3 Endpoint study record: Key Study_Dow_2005_Combined Repeated exposure_HET-DR-0306-9270-009 UUID: IUC5-c60d2a48-df7a-4a52-8a81-183e3264fada
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Section: 7.5.3 Endpoint study record: Key Study_Dow_2005_Combined Repeated exposure_HET-DR-0306-9270-009 UUID: IUC5-c60d2a48-df7a-4a52-8a81-183e3264fada
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: inhalation study

Reproductive function / performance (P0)

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

Details on results (P0)

See Section: 7.5.3
Endpoint study record: Key Study_Dow_2005_Combined Repeated exposure_HET-DR-0306-9270-009
UUID: IUC5-c60d2a48-df7a-4a52-8a81-183e3264fada

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
600 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

There were no treatment-related effects on litter size, pup body weights or sex ratio. Female pup body weights in the 200 ppm group were statistically identified as increased on day 1 postpartum. This increase was not dose related or associated with any other toxicologic effects and, thus, was considered spurious and unrelated to treatment.

Pup survival was slightly, but statistically significantly decreased in the 600ppm group on postnatal days 1 and 4 (96.9% and 94.4% respectively, control survival was 100% on both days). This finding was not considered to be toxicologically significant as the values for both day 1 and 4 were within the laboratory’s historical control range (96.2-100% and 94.0-100% respectively). In addition, there were no associated effects on litter size or other reproductive endpoints that would be indicative of a reproductive effect.

Effect levels (F1)

Dose descriptor:
NOAEC
Generation:
F1
Effect level:
600 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on these results, the no-observed-effect concentration for reproductive effects was 600 ppm, the highest concentration tested.