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Toxicological information

Toxicity to reproduction

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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: The study was conducted in a GLP facility to OECD guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
The purity of the test substance was determined by gas chromatography with flame ionization detection (GC/FID) to be 99.9% prior to use on the study and 99.8% at study termination. Based on these data, the test substance was considered to be stable during the test period. The structure
of the test substance was confirmed using mass spectrometry. The mass spectrum of the test substance was consistent with published spectra for this substance.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Twelve male and 12 female (nulliparous and non-pregnant) Sprague-Dawley rats [Crl:CD(SD)IGS BR] obtained from Charles River Laboratories, Stone Ridge (Kingston), NY were randomly assigned to each exposure group. The male and female rats were 63 or 66 days of age and weighed 324 ± 9 or 226 ± 9 grams (mean± SD), respectively, at the start of the study. Rats were chosen for these study because they are a common representative species for inhalation and developmental toxicity studies. They have a high fecundity and this strain is routinely used in rodent reproductive and developmental toxicity studies in our laboratory. Also, the rat is one of the two primary rodent species recommended for use in developmental toxicity
studies in the OECD Test Guideline

Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited vivarium in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). During nonexposureperiods, rats were singly housed in stainless-steel, wire-mesh cages in a room separate from the exposure room. No other study was housed in the same room as this study. Exposure cages were washed daily. Housing cages and racks were washed once a week. Absorbent paper, used to collect excreta, was changed daily.

The study room was maintained at 21.0- 24.0°C and 43.5- 68.5% relative humidity. A photoperiod of 12 hours light from 6 a.m. to 6 p.m. was maintained. Animals were isolated upon arrival and allowed to acclimate for a period of at least five days prior to assignment to this study. Animals were judged to be healthy prior to testing. Certified Rodent Diet [Purina Rodent Chow #5002, meal (P~ Feed, Inc. Richmond, IN] was available ad libitum except during exposure. Feed containers were cleaned and refilled at least once a week. No known contaminants which would interfere with the
outcome of this study were present in the feed. Water was available ad libitum, except during exposure, through an automatic watering
system. The source of the water was the local public water system. There have been no contaminants identified in periodic water analyses that would be expected to interfere with the conduct of the study. Upon arrival, all rats were identified by uniquely-numbered metal ear tags. Ear tags
which were lost during the study were replaced. During randomization, study-specific animal numbers were assigned to each animal. Cage cards, color-coded for each group, contained the study-specific animal number and the ear tag number.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Exposure concentrations were selected based on test results of a four-day probe study during which 3 male and 3 female rats per group were exposed to 5.0, 2.5, 1.0, or 0.0 mg/L of the test substance 6 hours/day for four days. All exposure conditions were as· described below. Significant toxicity was observed (minimal to minor reductions inactivity during exposure for the 5.0 mg/L group, reduced fecal volumes on Day 2 for
the 5.0 mg/L female rats, lower mean feed consumption between Days 0 and 1 for the 2.5 and 5.0 mg/L female rats) suggesting that these exposure concentrations could be tolerated during the main study without confounding interpretation of the study results, particularly with respect to pregnant animals. Therefore, exposure concentrations of 5.0, 2.5, 1.0, or 0.0 mg/L were selected for the reproduction/developmental toxicity screening
test.

The inhalation exposures were conducted in 590 L stainless-steel and glass inhalation chambers at target vapor concentrations of 5.0, 2.5, 1.0, and 0.0 mg/L. Animals were singly housed during the 6-hour exposures. The exposure chambers were maintained under negative pressure relative to room air. The air flow, temperature, and humidity were recorded approximately every 30 minutes. Chamber vapor concentrations were recorded at least once each hour. The test atmosphere was generated by metering the test substance into glass distillation columns packed with glass beads. Filtered, compressed air was passed through the glass bead-packed columns to evaporate the test substance. The test substance delivery rate and air flow rate were adjusted to produce the desired chamber target vapor concentration. The resultant vapor was directed via glass tubing to a tee just upstream of the inhalation chamber where it was mixed with filtered, conditioned outside air to produce a total airflow of 116 to 212 Lpm (12 to 22 air changes per hour). A Micro Laser Particle Counter (model JlLPC-301, Particle Measuring Systems, Inc., Boulder, CO) was used to measure the number and size of particulates in the chamber. The results indicated that an aerosol of the test substance was not present.

Once each week (Days 1, 8, 15, 22, 29, 36, 43, and 49), samples of chamber test atmosphere were collected into Tedlar bags. These samples were analyzed using a GC/FID. Daily MIRAN Vapor Concentration Determination Chamber vapor concentrations were monitored with a multipositional air sampling and analysis system. The system consisted of a single :MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) and a computer-operated four-port sampling valve (Valco Instruments, Houston, TX).Chamber vapor samples were continuously collected from each chamber through TEFLON tubing (0.48 mrn i.d.). The valve position was periodically changed to sample from each chamber at least once each hour. The voltage output of the MIRAN and chamber concentrations were printed in real-time and captured on electronic media. Voltage data were converted to concentration by linear interpolation between the calibration data points immediately on each side of the sampled data. A test to determine variations in concentration at different positions within the exposure chambers was conducted prior to study initiation. The air from the breathing zones of cage positions 1, 3, 7, 9, 14, 19, and 21 was sampled and compared to the concentration at a fixed reference position (cage 15). Based on deviations from the reference position of less than 10%, the chamber atmosphere was considered to be homogeneous.

Total chamber air flow was a combination of filtered, compressed air, which was used to vaporize the test substance and to carry the vapor from the generation system to the inhalation chamber, and additional filtered, compressed air dilution air. The air flow rate was continuously monitored using calibrated flowmeters (Gilmont Instruments, Barrington, IL). The oxygen content of the chamber exposure atmosphere was measured during exposure from the reference position using a MAS Mini OX Monitor (MAS Instrument Division, Pittsburgh, PA). The oxygen content of the chamber exposure atmosphere was~ 20%. Chamber temperature and humidity were measured using wet/dry bulb hygrometers and were recorded approximately every 30 minutes during exposure.
Details on mating procedure:
Male and female rats were mated 1:1 within the same dose group for 1 to 14 days. The study and ear tag numbers of the mated pairs were recorded. Copulation was verified through identification of sperm in vaginal smears or by appearance of a cqpulation plug. The morning of the day copulation was verified was considered Day 0 of gestation. Following copulation, the male and female rats were separated and housed individually until study termination. For female rats that showed no evidence of mating, the expected delivery date was calculated from the last day of possible copulation with a male.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Once each week (Days 1, 8, 15, 22, 29, 36, 43, and 49), samples of chamber test atmosphere were collected into Tedlar bags. These samples were analyzed using a GC/FID. Chamber vapor concentrations were monitored with a multipositional air sampling and analysis system. The system consisted of a single :MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) and a computer-operated four-port sampling valve (Valco Instruments, Houston, TX).
Chamber vapor samples were continuously collected from each chamber through TEFLON tubing (0.48 mrn i.d.). The valve position was periodically changed to sample from each chamber at least once each hour. The voltage output of the MIRAN and chamber concentrations were printed in real-time and captured on electronic media. Voltage data were converted to concentration by linear interpolation between the calibration data points immediately on each side of the sampled data.
Duration of treatment / exposure:
35-48 days for females (depending on the time for mating) and 51 days for males.
Frequency of treatment:
Daily
Details on study schedule:
The study consisted of four phases: pre-mating (14 days); mating (1 to 14 days); pregnancy (21 to 22 days); and early lactation (4 days). The female rats were exposed, 6 hours/day, through Day 19 of gestation (approximately 35-48 consecutive exposures). Females that delivered a litter, and their offspring, were euthanatized on Days 4 postpartum. Females showing no evidence of copulation were euthanatized 23 days after the last day of the mating period. Male rats were exposed, 6 hours/day, throughout the entire study (until all dams had littered or had been euthanatized due to non-delivery of a litter) for a total of 51 consecutive exposures. All male rats were euthanatized the following day. Mating male and female rats were mated 1: 1 within the same dose group for 1 to 14 days. The study and ear tag numbers of the mated pairs were recorded. Copulation was verified
through identification of sperm in vaginal smears or by appearance of a cqpulation plug. The morning of the day copulation was verified was considered Day 0 of gestation. Following copulation, the male and female rats were separated and housed individually until study termination. For female rats that showed no evidence of mating, the expected delivery date was calculated from the last day of possible copulation with a male.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5 mg/l
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
2.5 mg/l
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0 mg/l
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1 mg/l
Basis:
analytical conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
The study consisted of four phases: pre-mating (14 days); mating (1 to 14 days); pregnancy (21 to 22 days); and early lactation (4 days). The female rats were exposed, 6 hours/day, through Day 19 of gestation (approximately 35-48 consecutive exposures). Females that delivered a litter, and their offspring, were euthanatized on Days 4 postpartum. Females showing no evidence of copulation were euthanatized 23 days after
the last day of the mating period. Male rats were exposed, 6 hours/day, throughout the entire study (until all dams had littered or had been euthanatized due to non-delivery of a litter) for a total of 51 consecutive exposures. All male rats were euthanatized the following day.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
Male and female rats were mated 1:1 within the same dose group for 1 to 14 days. The study and ear tag numbers of the mated pairs were recorded. Copulation was verified through identification of sperm in vaginal smears or by appearance of a cqpulation plug. The morning of the day copulation was verified was considered Day 0 of gestation. Following copulation, the male and female rats were separated and housed individually
until study termination. For female rats that showed no evidence of mating, the expected delivery date was calculated from the last day of possible copulation with a male.
Sperm parameters (parental animals):
On the day of necropsy, motility was determined for the right epididymis. The epididymis was placed in a petri dish containing 1% bovine serum albumin in phosphate buffered saline pre-warmed to ~38°C. The epididymis was pierced three times with a scalpel blade followed by a period of at least 3 minutes to allow the sperin to swim out. A sample of the mixture in the petri dish was then loaded into the pre-warmed stage of the Hamilton Thorne IVOS automated sperm analyzer. Five fields were automatically selected by the analyzer and each motion image was recorded and stored on the optical disk. The images were subsequently analyzed and the percent motility was determined for each animal.

Testicular homogenization-resistant spermatid head counts were performed on the frozen left testes. After the tissue was thawed, the tunic was removed from the testis, the tissue was weighed, homogenized, and then vortexed. A 100 ul sample was transferred to a violet reaction vial containing a dye which uniquely stains the head of the sperm. A sample of stained sperm was placed into a 20 um deep glass slide which was loaded into
the Hamilton Thome IVOS automated sperm analyzer. Twenty fields were automatically selected by the analyzer for each animal and total sperm counts were determined. Head counts were reported on an absolute and relative (to testes weight) basis. Epididymal homogenization-resistant spermatozoan counts were performed on the frozen left epididymides. The epididymal tissue was processed in a similar manner as with the testicular tissue, except that the caudal left epididymis was used. Head counts were reported on an absolute and relative (to epididymal weight) basis.
Postmortem examinations (parental animals):
Adult male rats were fasted overnight, anesthetized with carbon dioxide and exsanguinated by severing the posterior vena cava on Day 51. Adult female rats that showed no evidence of delivery of a litter were euthanatized on Day 23 or 24 of the gestation phase. All other adult female rats were anesthetized with carbon dioxide and exsanguinated by severing the posterior vena cava on Day 4 post-partum. Following exsanguination, the adult animals were weighed and subjected to a necropsy and gross examination. Special attention was paid to the organs of the reproductive system. The uteri from all adult female rats were examined, and implantation sites were counted. The following tissues were collected from adult animals and fixed in 10% buffered formalin: ovaries, vagina, uterus, fallopian tubes, and male accessory sex glands, and gross lesions. The right testis and right epididymis (after motility analysis) were fixed in Bouin's fixative; after approximately 24 hours, these tissues were rinsed twice with 50% ethyl alcohol then stored in 70% ethyl alcohol. The left testis and left epididymis were placed into individual containers, frozen, with dry ice, and stored at -70°C.
Statistics:
Mean values were calculated for time-weighted average atmospheric concentration, chamber temperature, chamber relative humidity, chamber airflow, chamber nominal concentration, adult body weight and body weight change, male pup and female pup body weight and body weight change, feed consumption, organ weights, organ-to-body weight ratios, gestation period, litter size, and pup percent survival. Homogeneity of adult body weight, feed consumption, and organ weight data were evaluated using Bartlett's test (p < 0.01). Adult body weight, feed consumption, and organ weight data were evaluated using a one-way analysis of variance (ANOVA) (p < 0.05) and Duncan's multiple range test (p < 0.05). Adult body weight change, male pup and female pup body weight and body weight change, gestation period, litter size, and pup percent survival were evaluated using Bartlett's test (p < 0.01), one-way analysis of variance
(ANOVA) (p < 0.05), and Dunnett's t-test (p < 0.05) to indicate statistical significance (MINITAB Statistical Software, State College, PA). When the variances of the means were not considered equal by the Bartlett's test (p <0.01), the data were evaluated using a Kruskal-Wallis H-test (p < 0.05) followed by Mann-Whitney U-test (p < 0.05) (MINITAB Statistical Software, State College, PA). The reproductive performance of the dams was evaluated in a contingency table, using a Chi-square test (p <0.05).

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
minimal reductions in activity level at 5 mg/l only. Reduced activity is defmed as less movement, decreased alertness, and slower response to tapping on the chamber wall compared with activity levels exhibited by control animals.
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 examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

During exposure, the adult male and female rats exposed to 5.0 mg/L had minimal reductions in activity level. Reduced activity is defmed as less movement, decreased alertness, and slower response to tapping on the chamber wall compared with activity levels exhibited by control animals. All other adult animals appeared normal during exposure. No other test substance-related clinical abnormalities were observed. Incidental lesions consisted of alopecia, red discoloration of urine, superficial skin wounds, eschar formation, malocclusion, ocular or nasal porphyrin discharge, red discoloration of hair, and sialorrhea. One 2.5 mg/L male rat (#526) had severe hematuria, detected using a Multistix (a clinical dipstick designed for evaluation of urine samples), from Day 10 through study termination. At necropsy, the kidneys from Rat #526 appeared larger than normal. Microscopic evaluation of this animal's kidneys revealed hydronephrosis~ prominent granular deposits within the renal pelvis, as well as a focus of transitional epithelial mineralization.

Mean feed consumption values for all test substance-exposed male and female groups were comparable to those of the control groups throughout the study.

Mean body weights and body weight changes for all adult male test substance-exposed groups and mean body weights for all adult female exposed groups were comparable to those of the control groups throughout the study. The mean body weight change, between Days 0 and 14 of the pre-mating phase for the 1.0 mg/1 adult female group, was higher (p < 0.05) when compared with the control group. All other mean body weight changes for adult female exposed groups were comparable to those of the control group. The mean absolute, but not relative, epididymides weight for the 5.0 mg/l adult male group was higher (p < 0.05) when compared with the control group. Mean terminal body weights for adult male and female test substance-exposed groups and the mean absolute and relative testes weights for adult male exposed groups were comparable to those of the control group. No test substance-related lesions were observed at necropsy or on microscopic examination of collected tissues. See the pathologist's report beginning on page 60 for details of the gross and histopathology examinations.

Effect levels (P0)

Dose descriptor:
NOEC
Effect level:
2.5 mg/L air
Sex:
male/female
Basis for effect level:
other: NOEC was based upon reduced activity at 5 mg/l

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:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

For all examined parameters (rc~productive performance; gestation length; pup survival; prenatal loss; numbers of implants, live and dead pups, and male and female pups; and .pup body weight and pup body weight change), 1he test substance-exposed groups were comparable to the control
group. Abnormalities were observed in pups from all groups. These abnormalities included bruises under the skin, superficial skin wounds, bleeding from the toes, dried blood on the skin of the toes, eschar formation, purple tinge to the skin tone, and little or no milk in the stomach.
Additionally, pups were occasionally missing (presumably cannibalized) or found dead. These observations were distributed across all groups, and were not considered to be treatment-related.

Effect levels (F1)

Dose descriptor:
NOEC
Generation:
F1
Effect level:
5 mg/L air
Sex:
male/female
Basis for effect level:
other: The reproductive NOEC was based on no findings at the highest dose concentration in the P of F1 animals.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Exposure to the test substance resulted in reduced activity during exposure, for animals exposed to 5.0 mg/L. However, no adverse effects on reproduction or development were observed. Therefore, the no-observed-effect level (NOEL) for toxicity to adult animals was 2.5 mg/L and the NOEL for reproductive and developmental toxicity was determined to be 5.0 mg/L.
Executive summary:

The potential toxicity of the test substance was evaluated using a reproduction/developmental toxicity screening test. The test consisted of four phases: pre-mating (14 days), mating (1 to 14 days), gestation (21 to 22 days), and early lactation (4 days). Male and female Sprague-Dawley rats were exposed to concentrations of 5.0, 2.5, 1.0, or 0.0 mg/L of the test substance for six hours

per day, 7 days per week for a total of 35 to 48 exposures for female rats (through Day 19 of gestation) and 51 exposures for male rats. Exposure concentrations were selected based upon the results of a 4 day probe study that demonstrated minimal to minor reductions in activity during exposure for the 5.0 mg/L group, reduced fecal volume on Day 2 for the 5.0 mg/L female group,

and lower mean feed consumption between Days 0 and 1 for the 2.5 and 5.0 mg/L female rats. Both the mean analytical (GC/FID) and mean daily time-weighted average (MIRAN) concentrations were within 1.0 % of the target concentrations. The temperature and relative humidity inside the chambers during exposure were 21.2- 24.0°C and 52- 83%, respectively. All adult animals survived to study termination. Adult animals were observed for signs of toxicity prior to exposure, once per hour during exposure, and 30 minutes to one hour after exposure. Beginning on Day 20 of gestation (during the non-exposure period), each female rat was

removed from its cage and examined at least once daily. Pups were observed once daily. During exposure, reduced activity was observed for the adult 5.0 mg/L group. Adult animals from the 2.5, 1.0, and 0.0 mg/L groups appeared normal during exposure. No test substance-related clinical signs were observed when parental animals were examined outside of the chambers. No test substance-related changes in mean feed consumption, body weights, or body weight change were observed. At study termination, animals were anesthetized with carbon dioxide and exsanguinated. No test substance-related changes in mean terminal body weight or in reproductive organ weights were observed. Mean sperm motility and mean epididymal spermatozoan and testicular spermatid counts were comparable among the groups. No test substance-related gross pathology was observed for adult animals from any group. No exposure related changes were observed during histological examination of the reproductive organs of any of the test substance-exposed animals. No reproductive or developmental toxicity was observed and no test substance-related clinical

abnormalities were observed for the pups. In conclusion, exposure to the test substance resulted in reduced activity during exposure for animals exposed to 5.0 mg/L. However, no adverse effects on reproduction or development were observed. Therefore, the no-observed-effect level (NOEL) for toxicity to adult animals was 2.5 mg/L and the NOEL for reproductive and developmental toxicity was determined to be 5.0 mg/L.