Registration Dossier

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
Study period:
2004-2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and reported standard OECD guideline study conducted according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid; clear/yellow
Details on test material:
The test article, polyethylbenzene bottom stream (PEB), was received from ABC Laboratories, Columbia, Missouri, on August 18, 2004, as follows:

- Name of test material (as cited in study report): PEB Blend
Lot no. 030005
CAS no. 68987-42-8
Batch no. TS-16672
Exp. date: 7/13/05
[WIL log no. 6271A]

- Composition of test material, percentage of components:
diethylbenzene:0.01%
1,3,5-triethylbenzene:6.20%
1,2,4-triethylbnzene:7.23%
cyclohexylbenzene:0.66%
diphenylmethane:20.45%
1,1'-diphenylethane:25.42%
1,2-diphenylethane (bibenzyl):7.98%
1,1'-diphenylpropane:2.42%

- Physical state: Clear, yellow liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CHarles River Laboratories, Raleigh, NC
- Age at study initiation: 10 wk
- Weight at study initiation: Males 322-390g, females 201-258g
- Fasting period before study: none
- Housing: Individually housedin steel wire mesh cages until mating then paired 1:1 male:female in the males home cage until mating success confirmed (presence of vaginal plug or sperm present in vaginal lavage sample ). Mated females transferred to plastic boxes with corncob bedding as nesting material.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 16 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 deg C
- Humidity (%): 50 ±20%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hr light/dark

IN-LIFE DATES: From: 2 December 2004 To: 25 May 2005

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Dosing solutions prepared weekly.
Evaluated for: Homogeneity, resuspension homogeneity and stability
Analysis done prior to study initiation and during study (weekly during first two weeks and monthyl thereafter) to verify concentrations at each dose level
Vehicle: 100% pure Mazola corn oil (ACH Food Companies, Inc., Memphis Tennessee)
Expiry dates: January, March and April 2006
Test article dosing preparations were analytically confirmed to be homogenous and were stable for 8 days under room temperature storage conditions.

Dose volume was 5 ml.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until succesful mating has taken place or up to a maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Gestation day 0
- After 14 days, if a female showed no evidence of succesful mating they were transferred to the standard maternity cages until scheduled euthenasia
- After successful mating each pregnant female was caged (how): Following positive evidence of mating, the males were housed in suspended
wire-mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed-O'Cobs®; The Andersons, Industrial Products Division, Maumee, Ohio). The nesting material is periodically analyzed by the manufacturer for contaminants.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
-Gas Chromatography (GC) with flame ionization detection (FID)

Prior to the initiation of dosing (December 1, 2004), duplicate samples (1 mL each) for homogeneity determination were collected from the top, middle and bottom strata of each test article formulation and one sample (1 mL) was collected from the middle stratum of the vehicle. In addition, duplicate samples (1 mL each) from the top, middle and bottom strata of each test article formulation and one sample (1 mL) from the middle stratum of
the vehicle were collected following 8 days of room temperature storage for resuspension homogeneity and stability determinations. Samples (1 mL each) for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) for the first 2 weeks of dose administration and on a monthly basis thereafter. An additional set of samples was collected from the preparations used for the
second week of dose administration to verify concentration due to a potential labeling error. The results of these analyses confirmed that each formulation was of the appropriate concentration, and that the animals received the correct dosing formulations.

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.
Duration of treatment / exposure:
Dosing done via a ball tipped dosing canula.
Males dosed daily on study days 0-36 (14 days prior to pairing through to 1 day prior to euthanasia for FOB assessed males or on the day of Euthenasia)
Females dosed daily on study days 0 - either 39 (non-mated females) or 52 (14 days prior to pairing through to lactation day 3 for the FOB assessed females or 4 for the rest).
Frequency of treatment:
Daily
Details on study schedule:
Study initiated on day 0
Cohabitation of males and females on day 14 until successful mating occurred, or until 14 days of cohabitation complete
Day 35 - Male FOB
Day 36 - male euthenasia
Females continued through to LD4 and Euthenised
Day prior to scheduled female euthanasia, FOB conducted.

Pups euthanised on LD4

Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
20 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
80 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
320 mg/kg bw.day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
Clinical Observations:
All rats observed twice daily for moribundity and mortality. Clinical observations recorded daily. Once prior to study initiation and weekly thereafter, rats observed outside the home cage for behavioural changes. Animals were observed at dosing and 1 hour afterwards for signs of overt toxicity.
Body weights and food consumption:
Recorded weekly for males and females until the start of gestation. Thereafter female body weights recorded on GD 0, 4, 7, 11, 17, 20 and LD 1 and 4. Non-pregnant females weighed weekly. Food consumption was recorded over the same intervals except during mating.
Parturition:
Pregnant rats observed twice daily for initiation and completion of parturition and signs of dystocia. On postnatal day 0 pups were sexed and examined for malformations and the number of stillborn and live pups were recorded. Gestation length calculated from the data at which parturition began.
Neurobehavioural Parameters:
Functional Observational Battery (FOB) observations recorded for 6 rats per sex per dose group during week 5 (Males) and on LD4 (Females) approximately 1 hour after dosing. The same technicians performed the study without knowledge of the group assignment in sound attenuated rooms with a white nise generator set at 70+/-10dB. Observations included home cage and handling, open field, sensory (startle response, forelimb and hindlimb extension, air righting reflex, tail pinch), Neuromuscular observations (hind limb foot splay, fore and hindlimb grip strength, rotarod performance) and physiological observations (catalepsy, body weight and body temp.). Locomotor activity was recorded after completion of FOB using a photobea activity system. Data collected during 5-minute epochs for a test duration of 60 minutes. Total motor activity was a combination of fine motor skills and ambulatory motor activity.
Oestrous cyclicity (parental animals):
not done
Sperm parameters (parental animals):
Not done
Litter observations:
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.

Litter Viability and Deaths:
Each litter was examined 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. A daily record of litter size was maintained.

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

Sex Determination:
Pups were individually sexed on PND 0 and 4
Postmortem examinations (parental animals):
Necropsy:
Males sacrificed following mating period (approx wk5). Females that delivered were sacrificed on LD4. NUmber of former implantation sites and corpora lutea were recorded. Femails failing to deliver were sacrificed on postmating day 25 (females without evidence of delivery) or post co-habitation day 25 (females without evidence of mating). Uteri were stained with 10% ammonia sulfide for detection of early implantation loss. Females with total litter loss were sacrificed within 24hrs of total loss.
The following organs weighed for all parental animals:
adrenal glands, brain, heart, kidneys, liver, lungs, spleen, thymus, thyroids with parathyroids, testes, epididymides, prostate, ovaries with oviduct, uterus.

39 tissues and all gross lesions were collected and fixed in formalin (10% neutral buffered) except for testis which were fixed in Bouin's solution.

Clinical Pathology:
Blood samples collected for hematology and serum chemistry from non-fasted rats (6/sex/group) at scheduled necropsy.

Histopathology:
Slides prepared for protocol specified tissues and stained with H&E, except for testis stained with PAS. Tissues from all control and high dose animals examined microscopically. Kidneys, liver and thyroids in males and thyroid and thymus from females also examined in the low and mid dose groups.
Postmortem examinations (offspring):
Intact offspring dying were necropsied using a fresh dissection technique including the heart and major vessels (Stuckhardt and Poppe, 1984). Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.
Statistics:
Refer to other information/methods section below.
Reproductive indices:
Male (female) Mating Index (%) = No. of males (Females) with evidence of mating (or confirmed pregnancy) / Total No. Of males (females) used for mating x 100

Male Fertility Index (%) = No. of males Siring a Litter / Total No. of males used for mating x 100

Male Copulation Index (%) = No. Of males Siring a Litter / No Of males with evidence of mating (or females confirmed pregnant) x 100

Female Fertility Index (%) = No. Of females with confirmed pregnancy / Total no. of females used for Mating x 100

Female Conception index (%) = No of females with confirmed pregnancy/ No of females with evidence of mating x 100
Offspring viability indices:
Mean Live Litter size: Total no of viable pups on PND 0 / No of littes with viable pups PND 0

Postnatal survival between birth and PND0 or PND 4 (% per litter) = Sum of (Viable pups per litter on PND 0 or PND4 / No of Pups born per litter) / No. of litters per Group x100

Postnatal Survival for all other intervals (% per litter) = Sum (Viable pups per litter at end of interval N / Viable pups per Litter at start of Interval N) / No. of litters per group x100

Where N = PND0-1 or 1-4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals in the control, 20, 80 and 320 mg/kg/day groups survived to the scheduled necropsies. At the daily examinations, an increase in the incidence of hair loss on the ventral abdomen and/or left hindlimb was observed in the 320 mg/kg/day group males and females. This finding was noted as early as study day 4 and was observed throughout the remainder of the study. At the time of dose administration, excessive pawing of the cage floor and/or walls was observed in the 320 mg/kg/day group males and females. The majority of the animals exhibited this finding during study days 20-23; however, one male and one female were noted with this finding on study day 3. These findings were not noted 1 hour following dose administration. One hour following dose administration, clear material on the forelimbs and/or around the mouth or nose and red material around the mouth was observed the 320 mg/kg/day group males and females. These findings were noted as early as study days 1 (red material) and 3 (clear material) and were observed throughout the remainder of the study. Clear material around the mouth was also noted in the 80 mg/kg/day group females 1 hour following dose administration, beginning on study day 9. These findings did not persist to the observations on the following day and were potentially due to taste aversion, not to systemic toxicity. Other clinical findings were noted infrequently, similarly in the control group and/or were not observed in a dose-related manner.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
All animals in the control, 20, 80 and 320 mg/kg/day groups survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
Mean body weight gains in the 320 mg/kg/day group males were reduced throughout the entire study; the differences from the control group values were statistically significant (p<0.01). As a result of the reduced mean body weight gains, mean body weights in these males were 6.5%-13.1% lower than those in the control group during study days 13-35 (statistically significantly, p<0.01). These reductions were attributed to the test article. Mean body weight gains in the 80 mg/kg/day group males were reduced throughout the entire study. The differences from the control group values were statistically significant (p<0.05 or p<0.01) during study days 7-13 through 21-27 and when the pre-mating period (study days 0-13) and entire treatment period (study days 0-35) were evaluated. Mean body weights in these males were similar to those in the control group during study days 0-13, but were 6.3%-8.3% lower during study days 21-35 (statistically significant, p<0.05 or p<0.01). These reductions were attributed to the test article. Mean body weight gains in the 20 mg/kg/day group males were slightly reduced compared to the control group values during study days 0-7, 7-13 and 21-27. The difference was statistically significant (p<0.05) during study days 21-27. These reductions resulted in slightly (not statistically significant) lower mean body weight gains when the pre-mating period (study days 0-13) and entire treatment period (0-35) were evaluated. However, the reductions in mean body weight gains in the 20 mg/kg/day group males were not of sufficient magnitude to result in statistically significant reductions in mean body weights. Because there were no indications of systemic toxicity in this group, the reductions in mean body weight gain were not considered adverse.

Females:
Pre-mating:
There were no test article-related effects on mean body weights or body weight gains in the 20, 80 and 320 mg/kg/day group females. The only noteworthy difference from the control group was a reduction in mean body weight gain in the 80 mg/kg/day group during study days 0-7. This decrease was due to three females that lost 5 g of body weight or more during this interval. The difference from the control group was not statistically significant and no similar reductions were observed at 320 mg/kg/day. Therefore, the reduction observed in the 80 mg/kg/day group females was not considered test article-related. This reduction in mean body weight gain was not of sufficient magnitude to result in substantial decrements in mean body weights.

Gestation:
Mean maternal body weight gain in the 320 mg/kg/day group was similar to that in the control group during gestation days 0-4. Mean body weight gains in this group were reduced during gestation days 4-7, 7-11, 14-17, 17-20 and when the entire gestation period (days 0-20) was evaluated; the differences from the control group were statistically significant (p<0.05 or p<0.01) during gestation days 4-7, 17-20 and 0-20. Mean body weights in the 320 mg/kg/day group females were lower than those in the control group throughout the majority of the gestation period (5.6%-10.1% during gestation days 7-20); the differences were statistically significant (p<0.05 or p<0.01) during gestation days 11-20. These reductions were attributed to the test article. Mean body weights and body weight gains in the 20 and 80 mg/kg/day groups were generally similar to those in the control group throughout gestation. None of the differences from the control group were statistically significant.

Lactation:
Mean body weight gain in the 320 mg/kg/day group was similar to the control group value during lactation days 1-4. However, the mean body weights in this group remained reduced on lactation days 1 and 4 following the test article-related reductions in mean body weight gain during gestation. The difference from the control group was statistically significant (p<0.05) on lactation day 4.

No test article-related effects on mean body weights and body weight gains were noted in the 20 and 80 mg/kg/day groups during lactation. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 320 mg/kg/day group males was similar to that in the control group during study days 0-7. During the remainder of the pre-mating period (study days 7-13), mean food consumption in these males was reduced; the difference from the control group was statistically significant (p<0.01, g/animal/day only). These reductions were considered test article-related. However, when the entire pre-mating period (study days 0-13) was evaluated, mean food consumption in the 320 mg/kg/day group males was similar to that in the control group. Food consumption in these males was also similar to the control group value during the post-mating period (study days 27-35). Mean food consumption in the 20 and 80 mg/kg/day group males was similar to that in the control group throughout the study. No statistically significant differences were observed.

Females: Pre-mating
Mean food consumption in the 20, 80 and 320 mg/kg/day group females was similar to that in the control group throughout the pre-mating period. The only statistically significant (p<0.05, g/animal/day only) difference in food consumption was a reduction in the 80 mg/kg/day group when the entire pre-mating period (study days 0-13) was evaluated. No dose-response relationship was evident. Therefore, these differences were not considered test article-related.

Gestation:
No test article-related effects on mean maternal food consumption were observed at any dosage level. The only statistically significant (p<0.05 or p<0.01) differences from the control group were increases in food consumption, evaluated as g/kg/day, in the 320 mg/kg/day group during gestation days 11-14 and 0-20. These increases were attributed to the lower mean body weights in this group during gestation.

Lactation:
Mean food consumption in the 20, 80 and 320 mg/kg/day groups was unaffected by the test article during lactation. None of the differences from the control group were statistically significant.

FOB observations:

Unaffected by test article administration. There were no statistically significant differences for the test article-treated groups when compared to the control group during study week 5 (males) or on lactation day 4 (females).

Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A slight decrease in the mean absolute and percent eosinophils in the 80 and 320 mg/kg/day group males was observed compared to the control group values. The mean percent eosinophils in the 320 mg/kg/day group females was also reduced compared to the control group value. The differences were statistically significant (p<0.05 or p<0.01). These decreases were considered to be potentially test article-related.
Other statistically significant (p<0.05) differences from the control group values did not occur in a dose-related manner and/or were of minimal magnitude. A slight decrease (p<0.05) in mean red blood cells in the 320 mg/kg/day group females was not accompanied by other hematologic changes, and the apparent decrease was considered spurious. A slightly decreased (p<0.05) mean hematocrit value in the 80 mg/kg/day group females was not observed in a dose-related manner; therefore, this decrease was not considered test article-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test article-related effects on serum chemistry.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
No test article-related effects on male and female reproductive performance were observed at any dosage level. Mating indices were 100.0%, 100.0%, 91.7% and 100.0% in the control, 20, 80 and 320 mg/kg/day groups, respectively. Fertility indices were 91.7%, 100.0%, 83.3% and 100.0% and copulation/conception indices were 91.7%, 100.0%, 90.9% and 100.0% in the same respective groups. No statistically significant differences were noted between the control and test article-treated groups. One male in the 80 mg/kg/day group did not mate, and mating by one male each in the control and 80 mg/kg/day groups did not result in a litter. One female each in the control and 80 mg/kg/day groups mated but did not produce litters, and one female in the 80 mg/kg/day group did not mate. At necropsy, these three females were determined to be nongravid by ammonium sulfide staining. One female in the 320 mg/kg/day group had evidence of mating but did not deliver; this female was determined to be gravid at necropsy.
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.

Details on results (P0)

Clinical Observations and Survival:
All animals in the control, 20, 80 and 320 mg/kg/day groups survived to the scheduled necropsies. At the daily examinations, an increase in the incidence of hair loss on the ventral abdomen and/or left hindlimb was observed in the 320 mg/kg/day group males and females. This finding was noted as early as study day 4 and was observed throughout the remainder of the study. At the time of dose administration, excessive pawing of the cage floor and/or walls was observed in the 320 mg/kg/day group males and females. The majority of the animals exhibited this finding during study days 20-23; however, one male and one female were noted with this finding on study day 3. These findings were not noted 1 hour following dose administration. One hour following dose administration, clear material on the forelimbs and/or around the mouth or nose and red material around the mouth was observed the 320 mg/kg/day group males and females. These findings were noted as early as study days 1 (red material) and 3 (clear material) and were observed throughout the remainder of the study. Clear material around the mouth was also noted in the 80 mg/kg/day group females 1 hour following dose administration, beginning on study day 9. These findings did not persist to the observations on the following day and were potentially due to taste aversion, not to systemic toxicity. Other clinical findings were noted infrequently, similarly in the control group and/or were not observed in a dose-related manner.

Body weight:
Males:
Mean body weight gains in the 320 mg/kg/day group males were reduced throughout the entire study; the differences from the control group values were statistically significant (p<0.01). As a result of the reduced mean body weight gains, mean body weights in these males were 6.5%-13.1% lower than those in the control group during study days 13-35 (statistically significantly, p<0.01). These reductions were attributed to the test article. Mean body weight gains in the 80 mg/kg/day group males were reduced throughout the entire study. The differences from the control group values were statistically significant (p<0.05 or p<0.01) during study days 7-13 through 21-27 and when the pre-mating period (study days 0-13) and entire treatment period (study days 0-35) were evaluated. Mean body weights in these males were similar to those in the control group during study days 0-13, but were 6.3%-8.3% lower during study days 21-35 (statistically significant, p<0.05 or p<0.01). These reductions were attributed to the test article. Mean body weight gains in the 20 mg/kg/day group males were slightly reduced compared to the control group values during study days 0-7, 7-13 and 21-27. The difference was statistically significant (p<0.05) during study days 21-27. These reductions resulted in slightly (not statistically significant) lower mean body weight gains when the pre-mating period (study days 0-13) and entire treatment period (0-35) were
evaluated. However, the reductions in mean body weight gains in the 20 mg/kg/day group males were not of sufficient magnitude to result in statistically significant reductions in mean body weights. Because there were no indications of systemic toxicity in this group, the reductions in mean body weight gain were not considered adverse.

Females:
Pre-mating:
There were no test article-related effects on mean body weights or body weight gains in the 20, 80 and 320 mg/kg/day group females. The only noteworthy difference from the control group was a reduction in mean body weight gain in the 80 mg/kg/day group during study days 0-7. This decrease was due to three females that lost 5 g of body weight or more during this interval. The difference from the control group was not statistically significant and no similar reductions were observed at 320 mg/kg/day. Therefore, the reduction observed in the 80 mg/kg/day group females was not considered test article-related. This reduction in mean body weight gain was not of sufficient magnitude to result in substantial decrements in mean body weights.

Gestation:
Mean maternal body weight gain in the 320 mg/kg/day group was similar to that in the control group during gestation days 0-4. Mean body weight gains in this group were reduced during gestation days 4-7, 7-11, 14-17, 17-20 and when the entire gestation period (days 0-20) was evaluated; the differences from the control group were statistically significant (p<0.05 or p<0.01) during gestation days 4-7, 17-20 and 0-20. Mean body weights in the 320 mg/kg/day group females were lower than those in the control group throughout the majority of the gestation period (5.6%-10.1% during gestation days 7-20); the differences were statistically significant (p<0.05 or p<0.01) during gestation days 11-20. These reductions were attributed to the test article. Mean body weights and body weight gains in the 20 and 80 mg/kg/day groups were generally similar to those in the control group throughout gestation. None of the differences from the control group were statistically significant.

Lactation:
Mean body weight gain in the 320 mg/kg/day group was similar to the control group value during lactation days 1-4. However, the mean body weights in this group remained reduced on lactation days 1 and 4 following the test article-related reductions in mean body weight gain during gestation. The difference from the control group was statistically significant (p<0.05) on lactation day 4.

No test article-related effects on mean body weights and body weight gains were noted in the 20 and 80 mg/kg/day groups during lactation. None of the differences from the control group were statistically significant.

Food consumption:
Males:
Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 320 mg/kg/day group males was similar to that in the control group during study days 0-7. During the remainder of the pre-mating period (study days 7-13), mean food consumption in these males was reduced; the difference from the control group was statistically significant (p<0.01, g/animal/day only). These reductions were considered test article-related. However, when the entire pre-mating period (study days 0-13) was evaluated, mean food consumption in the 320 mg/kg/day group males was similar to that in the control group. Food consumption in these males was also similar to the control group value during the post-mating period (study days 27-35). Mean food consumption in the 20 and 80 mg/kg/day group males was similar to that in the control group throughout the study. No statistically significant differences were observed.

Females: Pre-mating
Mean food consumption in the 20, 80 and 320 mg/kg/day group females was similar to that in the control group throughout the pre-mating period. The only statistically significant (p<0.05, g/animal/day only) difference in food consumption was a reduction in the 80 mg/kg/day group when the entire pre-mating period (study days 0-13) was evaluated. No dose-response relationship was evident. Therefore, these differences were not considered test article-related.

Gestation:
No test article-related effects on mean maternal food consumption were observed at any dosage level. The only statistically significant (p<0.05 or p<0.01) differences from the control group were increases in food consumption, evaluated as g/kg/day, in the 320 mg/kg/day group during gestation days 11-14 and 0-20. These increases were attributed to the lower mean body weights in this group during gestation.

Lactation:
Mean food consumption in the 20, 80 and 320 mg/kg/day groups was unaffected by the test article during lactation. None of the differences from the control group were statistically significant.

FOB observations:

Unaffected by test article administration. There were no statistically significant differences for the test article-treated groups when compared to the control group during study week 5 (males) or on lactation day 4 (females).

Clinical pathology:
Hematology:
A slight decrease in the mean absolute and percent eosinophils in the 80 and 320 mg/kg/day group males was observed compared to the control group values. The mean percent eosinophils in the 320 mg/kg/day group females was also reduced compared to the control group value. The differences were statistically significant (p<0.05 or p<0.01). These decreases were considered to be potentially test article-related.
Other statistically significant (p<0.05) differences from the control group values did not occur in a dose-related manner and/or were of minimal magnitude. A slight decrease (p<0.05) in mean red blood cells in the 320 mg/kg/day group females was not accompanied by other hematologic changes, and the apparent decrease was considered spurious. A slightly decreased (p<0.05) mean hematocrit value in the 80 mg/kg/day group females was not observed in a dose-related manner; therefore, this decrease was not considered test article-related.

Serum Chemistry:
There were no test article-related effects on serum chemistry.

Pathology:
Macroscopic examinations:
No test article-related internal findings were observed at any dosage level in females that failed to deliver, the female with total litter loss or males and females at the scheduled necropsy. Macroscopic findings observed in the test article-treated groups occurred infrequently and/or in a manner that was not dose-related.

Organ Weights:
Test article-related increases in mean absolute and relative (to final body weight) liver and kidney weights were observed in the 80 and 320 mg/kg/day group males; mean absolute and relative liver and thyroid/parathyroid gland weights were increased in the 320 mg/kg/day group females. The majority of the differences from the control group was statistically significant (p<0.05 or p<0.01). Mean absolute and relative thymus gland weights in the 320 mg/kg/day group males and females were decreased (not statistically significant) compared to the control group values. These reductions were also considered test article-related.
Other statistically significant (p<0.05 or p<0.01) changes in organ weights included the following. Mean absolute brain weight was decreased in the 320 mg/kg/day group females. Mean relative weights were increased for the kidneys in the 20 mg/kg/day group males, left and right testis in the 80 and 320 mg/kg/day group males, brain in the 320 mg/kg/day males and adrenal gland in the 320 mg/kg/day group females. These changes were attributed to the test article-related decreases in mean body weights and were not considered direct effects of the test article.

Microscopic evaluation:

In the 320 mg/kg/day group, test article-related histopathologic alterations were observed in the male kidneys, liver and thyroid glands and in the female thyroid and thymus glands. In the males, increased incidences and severity of mineralization, multifocal deposits or irregular basophilic material, typically at the cortico-medullary junction, were noted in the kidneys, corresponding to increased kidney weights for these males. Two instances of unilateral minimal mineralization were observed in control group males, while primarily bilateral, mineralization of minimal to mild severity occurred in the 320 mg/kg/day group males. Hepatocellular hypertrophy was observed in the livers of the majority of the 320 mg/kg/day group males and corresponded to increased liver weights; in these males, hepatocytes were enlarged, and sinusoids were less prominent. Also in the 320 mg/kg/day group, seven of the males and five of the females had thyroid glands with smaller follicles and/or taller follicular epithelium than in the control group animals; both of these findings were diagnosed as follicular cell hypertrophy. The presence of follicular cell hypertrophy corresponded to increased thyroid gland weights in these males and females. Additionally, three females in the 320 mg/kg/day group had minimal to moderate atrophy of the thymus gland. This finding was not observed in the control group and contributed to the decreased mean thymus weight for females in the 320 mg/kg/day group. Therefore, atrophy of the thymus gland in these females was considered test article-related.

None of the histopathologic alterations observed in the 320 mg/kg/day group were observed in the 20 or 80 mg/kg/day group males and females. All other microscopic changes were consistent with normal background lesions in clinically normal animals of the strain and age used in this study, and were considered to be spontaneous and/or incidental in nature and unrelated to test article administration.

Reproductive Performance:

No test article-related effects on male and female reproductive performance were observed at any dosage level. Mating indices were 100.0%, 100.0%, 91.7% and 100.0% in the control, 20, 80 and 320 mg/kg/day groups, respectively. Fertility indices were 91.7%, 100.0%, 83.3% and 100.0% and copulation/conception indices were 91.7%, 100.0%, 90.9% and 100.0% in the same respective groups. No statistically significant differences were noted between the control and test article-treated groups. One male in the 80 mg/kg/day group did not mate, and mating by one male each in the control and 80 mg/kg/day groups did not result in a litter. One female each in the control and 80 mg/kg/day groups mated but did not produce litters, and one female in the 80 mg/kg/day group did not mate. At necropsy, these three females were determined to be nongravid by ammonium sulfide staining. One female in the 320 mg/kg/day group had evidence of mating but did not deliver; this female was determined to be gravid at necropsy.
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.

Gestation length and Parturition:
Mean gestation length in the 320 mg/kg/day group (22.6 days) was increased compared to the concurrent control group value (21.6 days). The difference was statistically significant (p<0.01), and the value was above the maximum mean value in the WIL historical control data (22.3 days). In the 320 mg/kg/day group, no females delivered on gestation day 21, five each delivered on gestation days 22 and 23 and one delivered on gestation day 24. In contrast, four and seven females in the control group delivered on gestation days 21 and 22, respectively. Therefore, the increase in gestation length in the 320 mg/kg/day group was considered test article-related. The female that delivered on gestation day 24 had a single pup that was found dead on PND 0. Mean gestation lengths in the 20 and 80 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

Effect levels (P0)

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Dose descriptor:
NOAEL
Remarks:
Systemic
Effect level:
20 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: based on decrements in body weight gains and/or food consumption and changes in organ weights in the 80 and 320 mg/kg/day groups and microscopic findings in the 320 mg/kg/day group
Dose descriptor:
NOAEL
Remarks:
Reproductive
Effect level:
20 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Based on the extended mean gestation length and non-statistically significant decreased mean number of implantation sites in the 320 mg/kg/day group and decreased numbers of pups born and live litter size on PND 0 in the 80 and 320 mg/kg/day groups

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:
not examined
Histopathological findings:
not examined

Details on results (F1)

PND 0 LITTER DATA AND POSTNATAL SURVIVAL

The mean numbers of pups born and live litter sizes on PND 0 in the 80 and 320 mg/kg/day groups were reduced compared to the control group values. Although the differences from the control group were not statistically significant, the reductions were attributed to the test article. The decreases in the 320 mg/kg/day group were due to female nos. 69985 and 70003 that delivered only one and seven pups, respectively. Female no. 69985 also had total litter loss of its abnormally small litter (one pup) on PND 0, which resulted in decreased (not statistically significant) mean postnatal survival
on PND 0 and from birth to PND 4. Because the decreases in mean postnatal survival were due to a single female with an abnormally low litter size/survival, these decreases were not considered test article-related. The mean number of pups born and live litter size in the 20 mg/kg/day group and the percentage of males at birth in all test article-treated groups were similar to the control group values. Postnatal survival in the 20 and 80 mg/kg/day groups was unaffected by test article administration.

GENERAL PHYSICAL CONDITION AND MORTALITIES

The numbers of F1 pups found dead and/or missing, as well as the general physical condition of all F1 pups in this study were unaffected by test article administration. Pups that were found dead numbered one, two, zero and four in the control, 20, 80 and 320 mg/kg/day groups, respectively. One pup each in the control and 20 mg/kg/day groups was missing and presumed to have been cannibalized.

OFFSPRING BODY WEIGHTS

Mean male and female pup body weights and body weight changes in the 20, 80 and 320 mg/kg/day groups were unaffected by test article administration during PND 1-4. No statistically significant differences from the control group were noted.

NECROPSIES OF PUPS FOUND DEAD

The numbers of pups (litters) found dead during PND 0-4 numbered 1(1), 2(2), 0(0) and 4(3) in the control, 20, 80 and 320 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

SCHEDULED PUP NECROPSIES (PND 4)

No internal findings that could be attributed to parental test article administration were noted at the necropsy of pups euthanized on PND 4. Internal findings included a white area on the liver in pup no. 69969-01 in the 20 mg/kg/day group and a hemorrhagic ring around the iris in pup no. 69983-12 in the 320 mg/kg/day group. No other internal findings were noted.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
neonatal
Generation:
F1
Effect level:
320 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: highest dose level tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The objective of this study was to investigate the potential adverse effects of the test article when administered for a minimum of 28 days and to evaluate the potential of the test article to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

All animals survived to the scheduled necropsy; there were no test article-related macroscopic findings. Test article-related clinical findings included hair loss on the abdomen or hindlimbs at the daily examinations and excessive pawing of the cage surfaces at the time of dose administration in the 320 mg/kg/day group males and females, and clear or red material on the forelimbs or around the mouth or nose 1 hour following dose administration in the 80 mg/kg/day group females and/or the 320 mg/kg/day group males and females. The findings observed at the time of dose administration and 1 hour following dose administration did not persist to the next observation time point. Clear or red material was attributed to potential taste aversion to the test article and was not considered a sign of systemic toxicity.

Mean body weights and body weight gains in the 80 and 320 mg/kg/day group males were reduced (generally statistically significant). Mean food consumption in the 320 mg/kg/day male group was also statistically significantly lower than the control group during the second week of test article administration. However, when the overall 2-week pre-mating period was evaluated, food consumption in this group was similar to the control group. Although the food consumption decrease during the second week of test article administration was slight and transitory in this group, it was considered a test article-related effect as corresponding decrements in body weight gain were noted. Mean body weight gains in the 20 mg/kg/day group males were not adversely affected by test article administration. Food consumption in the 20 and 80 mg/kg/day group males was unaffected by test article administration. In contrast, mean pre-mating body weights, body weight gains and food consumption in the 20, 80 and 320 mg/kg/day group females were unaffected by test article administration. The only effects on female body weights were during gestation and lactation, when mean body weights and body weight gains in the 320 mg/kg/day group were generally reduced (occasionally statistically significant). Mean food consumption during gestation and lactation was unaffected by test article administration.

No test article-related effects were noted during the FOB evaluations at study week 5 (males) and lactation day 4 (females). No test article-related effects on locomotor activity were observed at any dosage level.

Statistically significant decreases in mean absolute and/or percent eosinophils in the 80 mg/kg/day group males and the 320 mg/kg/day group males and females were considered potentially test article-related. There were no other test article-related hematologic findings; serum chemistry parameters were unaffected by test article administration at all dosage levels.

Mean absolute and relative (to final body weight) liver and kidney weights were increased in the 80 and 320 mg/kg/day group males. Mean absolute and relative liver and thyroid gland weights were increased and mean absolute and relative thymus weights were decreased in the 320 mg/kg/day group females. The majority of the differences from the control group were statistically significant. Corresponding histopathologic alterations were observed in the 320 mg/kg/day group males and females. Increased incidence and severity of mineralization, multifocal deposits or irregular basophilic material in the kidneys of the 320 mg/kg/day group males corresponded to increased kidney weights. Hepatocellular hypertrophy in the liver of these males and follicular cell hypertrophy in the thyroid gland of the 320 mg/kg/day group males and females corresponded to increased liver (both sexes) and thyroid gland weights (females), respectively, in these animals. Atrophy of the thymus in three 320 mg/kg/day group females contributed to the decreased mean thymus weight for females in this group.

Reproductive performance (mating, fertility and copulation/conception indices) in the test article-treated groups was unaffected by test article administration. Mean gestation length in the 320 mg/kg/day group was increased (statistically significant) and the mean number of pups born and live litter size on PND 0 in the 80 and 320 mg/kg/day groups were decreased (not statistically significant) compared to the control group values. In the 320 mg/kg/day group, one female that delivered seven pups and another that delivered a single pup on gestation day 24 contributed to the lower mean number of pups.

Applicant's summary and conclusion

Conclusions:
Based on the extended mean gestation length and non-statistically significant decreased mean number of implantation sites in the 320 mg/kg/day group and decreased numbers of pups born and live litter size on PND 0 in the 80 and 320 mg/kg/day groups, a dosage level of 20 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of PEB when administered orally by gavage to rats.
The NOAEL for systemic toxicity was also considered to 20 mg/kg/day based on decrements in body weight gains and/or food consumption and changes in organ weights in the 80 and 320 mg/kg/day groups and microscopic findings in the 320 mg/kg/day group. The NOAEL for neonatal toxicity was 320 mg/kg/day, the highest level tested in this study.
Executive summary:

All animals survived to the scheduled necropsy. Test article-related increases in the incidence of hair loss on the ventral abdomen and/or hindlimb at the daily examinations, excessive pawing of cage surfaces at the time of dose administration and clear or red material on various body surfaces 1 hour following dose administration were observed in the 320 mg/kg/day group. An increased incidence of clear and/or red material around the mouth was also observed in the 80 mg/kg/day group females 1 hour following dose administration. The clear or red material was considered to be due to potential taste aversion to the test article, and not a sign of toxicity. The test article-related findings observed at the time of dose administration and 1 hour following dose administration did not persist to the next observation time point. No test article-related clinical findings were observed in the 20 mg/kg/day group males or females.

Mean body weights and body weight gains in the 80 and 320 mg/kg/day group males were reduced generally throughout the study. Mean food consumption in the 320 mg/kg/day male group was also lower than the control group during the second week of test article administration. However, when the overall 2-week pre-mating period was evaluated, food consumption in this group was similar to the control group. Although the food consumption decrease during the second week of test article administration was slight and transitory in this group, it was considered a test article-related effect as corresponding decrements in body weight gain were noted. No test article-related effects on mean body weights, body weight gains or food consumption were observed in females at any dosage level during the pre-mating period. During gestation and lactation, mean body weights and/or body weight gains in the 320 mg/kg/day group were reduced. No test article-related effects on mean body weights were noted in the 20 and 80 mg/kg/day groups during gestation or lactation. Mean food consumption in the 20, 80 and 320 mg/kg/day group females was unaffected by test article administration during the premating, gestation and lactation periods.

No test article-related effects on male and female mating, fertility and copulation/conception indices were observed at any dosage level. The mean number of days between pairing and coitus were unaffected by test article administration.

No test article-related effects on FOB parameters or locomotor activity were observed in the males during study week 5 or the females on lactation day 4.

Decreases in mean absolute and/or percent eosinophils were observed in the 80 mg/kg/day group males and the 320 mg/kg/day group males and females. There were no other test article-related hematologic findings; serum chemistry parameters were unaffected by test article administration at all dosage levels.

Mean gestation length in the 320 mg/kg/day group females was increased compared to that in the control group. One female in this group delivered a single pup on gestation day 24 that was found dead on the day of delivery.

There were no test article-related macroscopic findings at the scheduled necropsies. The mean numbers of implantation sites in the 80 and 320 mg/kg/day groups were decreased compared to the control group value. The mean number of unaccounted-for sites in the

320 mg/kg/day group was increased compared to that in the control group. No test article-related effects on these parameters were observed in the 20 mg/kg/day group.

Mean absolute and relative kidney weights in the 80 and 320 mg/kg/day group males and mean absolute and relative liver weights in the 320 mg/kg/day group males and females were increased. Also in the 320 mg/kg/day group, mean absolute and relative thyroid

gland weights were increased in the females, and mean absolute and relative thymus weights were decreased in males and females. No test article-related effects on organ weights were observed in the 20 mg/kg/day group males or females.

Test article-related increased incidences and severity of mineralization, multifocal deposits or irregular basophilic material were noted in the kidneys in the 320 mg/kg/day group males, correlating to increased kidney weights. Hepatocellular hypertrophy was

observed in the livers of these males, corresponding to increased liver weights. Follicular cell hypertrophy was observed in the thyroid gland in the 320 mg/kg/day group males and females. This finding correlated to increased thyroid gland weights in these animals.

Atrophy of the thymus was observed in three females in the 320 mg/kg/day group, corresponding to decreased thymus weight in these females. No test article-related microscopic findings were observed in the 20 or 80 mg/kg/day group males and females.

The mean number of pups born and live litter sizes on PND 0 were reduced in the 80 and 320 mg/kg/day groups. These parameters in the 20 mg/kg/day group were unaffected by test article administration. No test article-related effects on the percentage of males at

birth or postnatal survival were noted at any dosage level.

The general physical condition of the pups and mean pup body weights were unaffected by administration of the test article to the parental animals at all dosage levels. There were no test article-related necropsy findings in the pups that were found dead or at the

scheduled necropsy on PND 4.

Overall based upon the above reanalysis of the reproductive toxicity data and taking into account individual animal data,the following was concluded:

A. There is no treatment- related reproductive toxicity at the mid dose level (80 mg/kg/day) associated to the test material and

B. Treatment-related reproductive effects at the high dose level (320 mg/kg/day) are associated with significant parental systemic toxicity.

Therefore, the reproductive and developmental toxicity no-observed-adverse-effect level (NOAEL) for this study is 80 mg/kg/day (the mid dose); the reproductive and developmental toxicity lowest-observed-adverse-effect level (LOAEL) is 320 mg/kg/day and is accompanied by maternal systemic toxicity.

Overall,the effects observed in the OECD 422 study indicate that the registered substance exerts adverse effects on reproductive organs or tissues indirectly; these and other reported effects are associated with general systemic toxicity. Together outcome of OECD 422 study does not warrant the requirement for triggered EOGRTS testing pursuant to Annex IX, Section 8.7.3.