bis(diethylamino)-N,N-diethylmethaniminium chloride

Toxicity to reproduction

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: OECD 421 (1995) and EPA OPPTS 870.3550 (2000); GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sixty male and 60 female Crl:CD®(SD) rats were received from Charles River Laboratories, Inc., Raleigh, North Carolina, on October 19, 2004. The animals were approximately 56 days old upon receipt and 10 weeks old at initiation of test article administration. Each animal was examined by a qualified technician on the day of receipt and weighed 1 day after receipt . Male body weights ranged from 324.4 to 396.4 g and female body weights ranged from 215 to 248.3 g on the initial day of test article administration. Each rat was uniquely identified by an eartag displaying the animal number and housed for a minimum of 16 days for acclimation purposes. Following receipt, all F0 parental test animals were housed individually, except during the mating period, in clean, stainless steel wire-mesh cages suspended above cage-board. Individual cage cards displaying the animal number, group number, study number, dosage level and sex of the animal were affixed to each cage. The cage-board was changed three times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy of the parental generations, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study. All animals were housed throughout the acclimation period and during the study in an environmentally controlled room. Actual mean daily temperature ranged from 70.3°F to 70.6°F (21.3°C to 21.5°C) and mean daily relative humidity ranged from 43.2% to 57.0% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

For the control group (Group 1), a sufficient amount of deionized water was dispensed into a labeled storage container. The vehicle was stirred continuously throughout dose administration. The test article formulations were weight/volume (test article/vehicle) mixtures. For the test article-treated groups, the appropriate amount of the test article for each group was weighed into a tared, calibrated storage container. Approximately 70% of the volume of vehicle was added to each container. The formulations were mixed until uniform using a magnetic stirrer. A sufficient volume of the vehicle was added to each container to bring the formulations to the calibration mark. The pH of the first preparation was 6.08-7.31 for the test article formulations and 8.44 for the vehicle. The test article formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored refrigerated. The test article formulations were stirred continuously throughout the preparation, sampling and dose administration procedures. The test article formulations were visually inspected by the study director at experimental start and were found to be visibly homogeneous and acceptable for dosing. The test article formulations were analytically confirmed to be homogeneous and were stable under the specified storage conditions.

Details on mating procedure:

The animals were paired on a 1:1 basis within each treatment group after 14 days of treatment (15 days for the two males paired with the replacement females). All animals were randomly selected for pairing, avoiding sibling matings. A breeding record containing the male and female identification numbers and the start date of cohabitation was prepared. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the initiation of dosing, duplicate samples (1 mL each) for homogeneity determination were collected from the top, middle and bottom strata of the test article formulations; a single sample was collected from the middle stratum of the vehicle control group. In addition, duplicate samples (1 mL each) for stability determinations were collected from the top and bottom strata of these same dosing formulations following 8 days of refrigerated storage. Samples (1 mL each) for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared for the first and third weeks of dosing. All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, LLC. The test article formulations were homogeneous, contained the amounts of test article specified in the protocol and were stable for at least 8 days under refrigerated conditions.

Duration of treatment / exposure:

Males: Two weeks prior to mating, during the two week mating period and approximately four and a half weeks after mating (for a total of 60 doses)
Females: Two weeks prior to mating, during the two week mating period and through lactation day 3

Frequency of treatment:

daily

Details on study schedule:

Males received 14 or 15 daily doses prior to mating. Males were dosed throughout the mating period through the day prior to euthanasia for a total of 60 doses. Females (including the two replacement animals) received 14 daily doses prior to pairing and were dosed through lactation day 3 (the day prior to necropsy) for a total of 39-43 doses; females that failed to deliver were dosed through the day prior to euthanasia for a total of 41 and 50 doses. The F1 pups were euthanized on lactation day 4.

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 150, and 300/250 mg/kg/day
Basis:

No. of animals per sex per dose:

12/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

A series of range-finding and acute studies have been conducted with the test substance. These studies were required for the selection of doses for a 28-day toxicity study conducted according to OECD Test Guideline 407. These studies were necessary because of the steep dose-response curve for the test substance (mortality occurring at doses just slightly greater than no effect doses) as well as a dimorphic response, with females showing greater toxicity than males. Based on these previous studies, the dosage levels selected for the 28-day oral toxicity study of the test substance conducted according to OECD Test Guideline 407 in rats were 25, 100 and 250 mg/kg/day. In that study, male and nonpregnant female rats received the test article in deionized water at dosage levels of 25, 100 or 250 mg/kg/day for 28 consecutive days. There were no test article-related effects on survival, body weights and food consumption. FOB, motor activity, hematology, serum chemistry and urinalysis parameters were unaffected by test article administration. There were no test article-related macroscopic or microscopic findings or effects on organ weights. Due to the lack of toxicity at 250 mg/kg/day, the high dosage of 300 mg/kg/day was selected in the current study to induce some signs of systemic toxicity in the F0 animals, although the range-finding data suggested this might exceed the tolerable dose. Dosage levels of 50 and 150 mg/kg/day were selected to evaluate potential dose-response relationships. Following the death of two females after the first 300 mg/kg dose, selection of a reduced high dosage was required. With mortality at 300 mg/kg from a single dose, and with pregnant females being dosed in the present study, the 250 mg/kg/day dosage that was used in the previous 28-day study was selected.

Examinations

Parental animals: Observations and examinations:

Observations and examinations: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. In addition, the animals were observed for appearance, behavior and signs of toxicity at the time of dose administration and within 1-2 hours after completion of dose administration. Detailed physical examinations were recorded weekly for all parental animals throughout the study period. At the time of these weekly observations, the animals were removed from their home cages and placed in a standard arena for observation of changes in gait, posture or clonic or tonic movements. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

Individual F0 male body weights were recorded on the first day of dose administration and weekly thereafter and prior to the scheduled necropsy. Individual F0 female body weights were recorded on the first day of dose administration and weekly thereafter until evidence of copulation was observed. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4.

Individual F0 male and female food consumption was measured weekly until pairing. Food intake was not recorded during the mating period. Following mating, male food consumption was measured on a weekly basis until the scheduled necropsy. Female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4.

Litter observations:

All females were allowed to deliver naturally and rear their young until euthanasia (PND 4). During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started. Females with total litter loss or that did not deliver were euthanized by carbon dioxide inhalation within 24 hours of litter loss or on post-mating day 25, respectively.

Each litter was examined twice daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition on PND 0. A daily record of litter size was maintained. Intact offspring dying from PND 0 to 4 were necropsied using a fresh dissection technique.

Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.

Pups were individually weighed and sexed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by group.

Pups were individually sexed on PND 1 and 4.

Postmortem examinations (parental animals):

A complete necropsy was conducted on all F0 animals found dead or at termination following euthanasia by carbon dioxide inhalation. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities, including viscera. The numbers of former implantation sites and corpora lutea were recorded for all F0 females, if possible. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss. The F0 tissues and organs that were collected and placed in 10% neutral-buffered formalin (except as noted) are listed below. Organs weights (see listed below) were also collected from all F0 animals at the scheduled necropsies. Except as noted, paired organs were weighed together.

Microscopic evaluations were performed on select tissues (see list below) for all F0 animals from the control and high dose groups. After fixation, protocol-specified tissues were trimmed according to standard operating procedures and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at 4-8 microns, mounted on glass microscope slides and stained with hematoxylin and eosin, except as noted.

Postmortem examinations (offspring):

All F1 pups were examined externally for gross abnormalities, euthanized on PND 4 via an intraperitoneal injection of sodium pentobarbital solution, and discarded without further examination.

Statistics:

Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Due to the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ by ±1 in the last significant figure. Data obtained from nongravid animals were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit. Parental mating, fertility, copulation and conception indices were analyzed using the Chi-square test with Yates’ correction factor. Mean parental (weekly, gestation and lactation) and offspring body weights and body weight changes, parental food consumption data, pre-coital intervals, gestation lengths, implantation sites, live litter sizes, unaccounted sites, numbers of pups born and absolute and relative organ weights were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p is less than 0.05) intergroup variance, Dunnett's test was used to compare the test article-treated groups to the control group. Mean litter proportions (percent per litter) of postnatal pup survival and the percentage of males per litter were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p is less than 0.05) intergroup variance, Dunn’s test was used to compare the test article-treated groups to the control group. Histopathological findings in the test article-treated groups were compared to the control group using a one-tailed Fisher’s Exact test.

Reproductive indices:

The following reproductive indices were calculated: Male (Female) Mating Index (%); Male Fertility Index (%); Male Copulation Index (%); Female Fertility Index (%): and Female Conception Index (%).

Offspring viability indices:

The following litter parameters were calculated: Mean Live Litter Size; Postnatal Survival Between Birth and PND 0 or 4; and Postnatal Survival for All Other intervals.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

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)

Two females, administered 300 mg/kg/day, were found dead on study day 1, following the first administration on study day 0. These females were replaced on study. Because of these deaths, the 300 mg/kg/day dosage level was lowered to 250 mg/kg/day on study day 1 for males and females (i.e. the first dose at this level was given on study day 1). A female (administered 250 mg/kg/day) in the 300/250 mg/kg/day group was found dead on gestation day 20; the cause of death could not be determined but was considered to be test article-related. On the day prior to death, this female had decreased defecation and red material around the nose. All other F0 animals in the control, 50, 150 and 300/250 mg/kg/day groups survived to the scheduled necropsy.

At the time of dose administration, salivation and/or clear material around the mouth were noted in all test article-treated groups. The incidence of these findings increased with dosage level, but the findings did not persist to 1 hour following dose administration at any dosage level in either sex, suggesting a local effect of the test article rather than a systemic response. The onset of clear material around the mouth and salivation was study day 0 and study day 5, respectively, and these findings were noted sporadically throughout the remainder of the study. One male in the 50 mg/kg/day group had a mass in the inguinal area; at necropsy, an inguinal herniation of the left testis and epididymis was noted. Therefore, the mass was not related to the test article. Other clinical findings noted in the test article-treated groups, occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.

Mean body weight gains in the 150 and 300/250 mg/kg/day group males were reduced compared to the control group value during the first week of dose administration. The differences were statistically significant and corresponded to slightly reduced food consumption (not statistically significant) in these males during the same interval. These reductions resulted in reduced mean body weight gains in the 150 and 300/250 mg/kg/day group males when the entire pre-mating period was evaluated. The differences from the control group were statistically significant. Mean male body weight gains in the 150 and 300/250 mg/kg/day group were generally similar to the control group values during the remainder of the study. No statistically significant differences were observed, and there were generally no dose-related or sustained trends when slight reductions were observed. When the entire treatment period was evaluated, mean male body weight gains in the 150 and 300/250 mg/kg/day groups were slightly (not statistically significant and without any clear dose response) reduced compared to the control group value. The reduced body weight changes at 150 and 300/250 mg/kg/day were transient, with no other indications of male toxicity; therefore, these reductions were not considered adverse. In addition, the deficits in mean male body weight gain in these groups were not of sufficient magnitude to result in any statistically significantly lower mean body weights throughout the study.

Slight reductions in mean female body weight gains in the 150 and 300/250 mg/kg/day groups were noted during study days 0-6. The reductions at 300/250 mg/kg/day coincided with the death of two females following the first dose administration at 300 mg/kg; however, the differences from the control group were not statistically significant or dose related, and there were no corresponding effects on food consumption during the same interval. Therefore, the relationship of these transient female body weight reductions at 300/250 mg/kg/day to test article administration was considered equivocal. The body weight effect at 150 mg/kg/day was transient, with no other maternal effects or indications of toxicity; therefore, the slight reduction in body weight change at 150 mg/kg/day was not considered adverse. Mean body weight gains in these females were similar to those in the control group during study days 6-13. When the premating period was evaluated, mean body weights and mean body weight gains in all groups of females were similar to those in the control group. Differences from the control group were slight and not statistically significant.

No test article-related effects on mean male and female body weights, body weight gains and cumulative body weight gains were noted in the 50 mg/kg/day group. None of the differences from the control group were statistically significant.

Mean maternal body weight gain in the 300/250 mg/kg/day group was similar to that in the control group during gestation days 0-4. A mean body weight loss was observed in this group during gestation days 4-7 compared to a gain in the control group. Five of 12 females in this group lost 2 to 31 g during this interval. Mean body weight gain in the 300/250 mg/kg/day group was slightly increased compared to the control group value during gestation days 7-11, reduced during gestation days 11-14 and 14-17 and similar to that of the control group during the remainder of gestation. When the entire gestational period was evaluated, mean body weight gain in the 300/250 mg/kg/day group was slightly reduced compared to the control group value. None of the differences at any interval were statistically significant compared to the control group. Mean maternal body weight changes during gestation in the 300/250 mg/kg/day group were sporadic and not statistically different from control group values; therefore, the relationship of these body weight changes to the test article was inconclusive. Mean body weights in the 300/250 mg/kg/day group were similar to the control group values throughout gestation.

Mean maternal body weights and body weight gains in the 50 and 150 mg/kg/day groups were unaffected by test article administration during gestation. Differences from the control group were slight and not statistically significant.

Mean maternal body weights and body weight gains in the 50, 150 and 300/250 mg/kg/day groups were unaffected by test article administration during lactation. Differences from the control group were slight and not statistically significant.

Mean food consumption, evaluated as g/animal/day, in the 150 and 300/250 mg/kg/day group males was slightly reduced compared to the control group value during study days 0-6. The difference was not statistically significant; however, the reduction corresponded with statistically significant reduction in mean body weight gain in these males during this interval. Mean food consumption in these males was similar to that in the control group throughout the remainder of the study and when the pre-mating and entire treatment periods were evaluated. No statistically significant differences were noted.

Mean food consumption in the 50 mg/kg/day group males and in the 50, 150 and 300/250 mg/kg/day group females was unaffected by test article administration. Differences from the control group were slight and not statistically significant.

Mean maternal food consumption, evaluated as g/animal/day, in the 300/250 mg/kg/day group was similar to that in the control group during gestation days 0-4 but reduced during gestation days 4-7, 11-14 and 14-17. The differences from the control group during gestation days 4-7 and 14-17 were statistically significant. A slight reduction (not statistically significant) in food consumption in this group was noted when the entire gestation period (days 0-20) was evaluated compared to the control group value. The reductions in the 300/250 mg/kg/day group corresponded to slight deficits in mean body weight change during these intervals. No other reductions in mean food consumption were observed in this group during gestation. Mean maternal food consumption in the 50 and 150 mg/kg/day groups was unaffected by test article administration during gestation. Differences from the control group were slight and not statistically significant.

Mean maternal food consumption in the 50, 150 and 300/250 mg/kg/day groups was unaffected by test article administration during lactation. Differences from the control group were slight and not statistically significant.

No test article-related effects on F0 reproductive performance were observed at any dosage level. Male and female mating indices were 100.0% in all groups, including the control group. Male and female fertility, male copulation and female conception indices were 91.7%, 100.0%, 91.7% and 100.0% in the control, 50, 150 and 300/250 mg/kg/day groups, respectively. No statistically significant differences were noted between the control and test article-treated groups. One mating pair each in the control and 150 mg/kg/day groups had evidence of mating but did not produce a litter. The mean numbers of days between pairing and coitus in the test article-treated groups were similar to the control group value. None of these differences were statistically significant.

No test article-related effects were noted on mean gestation lengths or parturition at any dosage level. Mean F0 gestation lengths in the test article-treated groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the 50, 150 and 300/250 mg/kg/day groups were 21.8, 21.5, and 21.8 days, respectively, compared to mean gestation lengths of 21.5 days in the concurrent control group and 21.8 days in the WIL historical control data. No signs of dystocia were noted at any dosage.

Two females in the 300/250 mg/kg/day group (found dead on study day 1, replaced on study) had no test article-related internal findings at necropsy. A third female in the 300/250 mg/kg/day group was found dead on gestation day 20. This female was internally normal with 14 fetuses with no apparent malformations in utero. All other animals survived to the scheduled necropsies.

At the scheduled F0 male and female necropsies, no test article-related internal findings were observed at any dosage level. Macroscopic findings observed in the test article treated groups occurred in single animals. One female each in the control and 150 mg/kg/day groups had evidence of mating, but did not deliver; both were nongravid.

No test article-related effects were observed on the number of former implantation sites and the number of unaccounted sites. The differences between the control and test article-treated groups were slight and not statistically significant.

No test article-related effects on organ weights (absolute, relative to final body weight and relative to brain weight) were observed at any dosage level in either sex when the test article-treated groups were compared to the control group. None of the differences from the control group were statistically significant.

No test article-related microscopic findings were observed in the 300/250 mg/kg/day group males (scheduled necropsy) and females (post-mating day 25, total litter loss and scheduled necropsy). All microscopic findings were considered to be spontaneous and/or incidental and unrelated to treatment. All females in the control and 300/250 mg/kg/day groups, except for one nongravid female in the control group, had changes in the reproductive tissues and mammary gland suggestive of recent parturition. Considering the physiological state of these females, those changes were considered normal.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Mortality, reduced body weight and food consumption at 300/250 mg/kg/day

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

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:

no effects observed

Details on results (F1)

Mean live litter size on PND 0 in the 300/250 mg/kg/day group was reduced due to one female with total litter loss (10 pups) on that day. It could not be determined whether or not these pups were born alive prior to being found dead. No milk was present in the stomachs of these pups. Due to the scope of this screening study, the number of litters available for evaluation (11 in the 300/250 mg/kg/day group) was limited. However, because total litter loss in rats is rare in control group females (only 13 litters lost of 1583 litters in the WIL historical control data), this total litter loss was considered test article-related. There was no effect on litter size for the 10 dams that had live litters in the 300/250 mg/kg/day group. Mean live litter sizes in the 50 and 150 mg/kg/day groups were similar to that in the control group. The mean number of pups born and the percentage of males per litter at birth were unaffected by the test article at all dosage levels. Differences from the control group were slight, were not statistically significant and/or did not occur in a dose-related manner.

Postnatal survival on PND 0 and from birth to PND 4 was reduced (not statistically significant) in the 300/250 mg/kg/day group. These decreases were due to one female in this group with total litter loss on PND 0. There was no effect on postnatal survival for pups in the other 10 litters in the 300/250 mg/kg/day group. Postnatal survival in this group was similar to that in the control group during PND 0-1 and 1-4. No test article related effects on postnatal survival were noted in the 50 and 150 mg/kg/day groups. Differences from the control group were slight and not statistically significant.

The numbers of F1 pups found dead in the 50 and 150 mg/kg/day groups, and the number of pups missing, as well as the general physical condition of all F1 pups in the 50, 150 and 300/250 mg/kg/day groups were unaffected by test article administration. Pups (litters) that were found dead numbered 1(1), 5(3), 3(2) and 11(2) in the control, 50, 150 and 300/250 mg/kg/day groups, respectively. One and two pups in the control and 50 mg/kg/day groups, respectively, were missing and presumed to have been cannibalized. The increase in the number of pups found dead in the 300/250 mg/kg/day group was due to one female with total litter loss (10 pups) on PND 0.

Mean male and female pup body weights and body weight gains during PND 1-4 in the 50, 150 and 300/250 mg/kg/day groups were unaffected by maternal test article administration. No statistically significant differences from the control group were noted. The numbers of pups (litters) found dead from PND 0 to 4 numbered 1(1), 5(3), 3(2) and 11(2) in the control, 50, 150 and 300/250 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration of the test article were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, the only other internal finding (small spleen) was observed in one control group pup; this pup also had edematous skin. No other internal findings were noted.

Effect levels (F1)

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Body Weight Changes (g)

Dose (mg/kg/day)	0	50	150	300/250
Males
Day 0-6
Mean	23.9	21.4	16.5*	16.0*
S.D.	6.11	6.73	6.51	6.42
N	12	12	12	12
Day 0-13
Mean	52.3	51.0	39.3**	41.3*
S.D.	10.94	9.26	11.12	6.80
N	12	12	12	12
* Significantly different from the control group at 0.05 using Dunnett’s test ** Significantly different from the control group at 0.01 using Dunnett’s test

Food Consumption During Gestation

Group (mg/kg/day)	0	50	150	300/250
Females
Day 4-7
Mean	22.	21.	21.	19.*
S.D.	3.1	1.2	1.9	3.4
N	11	12	11	12
Day 14-17
Mean	24.	23.	23.	20.**
S.D.	2.4	1.9	2.3	2.9
N	11	12	11	12
* Significantly different from the control group at 0.05 using Dunnett’s test ** Significantly different from the control group at 0.01 using Dunnett’s test

Applicant's summary and conclusion

Executive summary:

In the reproductive/developmental toxicity screening study, a test substance dosing of 300/250 mg/kg/day resulted in the death of two females following the first dose at 300 mg/kg/day and one female at gestation day 20 following reduction of the dose to 250 mg/kg/day starting on study day 1. Body weight and food consumption were reduced primarily during the first week of the pre-mating period in the 300/250 mg/kg/day group. A single animal in the 300/250 mg/kg/day dose group had total litter loss; no effects on live litter size or postnatal survival were observed in the litters of the other dams in this group. No test article-related adverse effects were observed at 50 and 150 mg/kg/day. The NOAEL for this OECD 421 study was considered to be 150 mg/kg/day.