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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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]

Coproduct of ethylbenzene manufacture and a Class II complex mixture consisting of various isomers of alkylbenzene and diphenyl hydrocarbons. Contains 15-32% Diphenylethanes, including 25.4% 1,1’-diphenylethane and 7.98% 1,2-diphenylethane (bibenzyl). CAS 68978-42-8.


- 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: 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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral 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
Analytical verification of doses or concentrations:
yes
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 euthenasia for Functional Observational Battery 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 Functional Observational Battery assessed females or 4 for the rest).
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
20 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
80 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12 males and females per dose
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
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.

Sacrifice and pathology:
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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
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.

Lactaition:
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.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Decreased body weight and/or food consumption, organ weight changes at 80 mkd and microscopic findings in organs at 320 mkd
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: decreased body weight and/or food consumption, organ weight changes

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for systemic toxicity in the OEDC 422 study was 20 mg/kg/day. Effects included decreased adult body weight and body weight gain, decreased 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. Neurobehavioral parameters were not affected by PEB Bottoms treatment.

Executive summary:

Sprague Dawley rats (12M, 12F per group) were given PEB Bottoms in corn oil at doses of 0, 20, 80 and 320 mg/kg/day by oral gavage, 7 days/week. Males were treated from 14 days prior to mating to 1 day prior to sacrifice or on the day of sacrifice for males assessed for neurobehavioral parameters, for a total of 37-39 days. Females were treated from 14 days prior to mating through gestation to lactation day (LD) 3 or 4 if assessed for neurobehavioral parameters for a total of 39 (non-mated females) to 52 doses.

All rats survived to scheduled necropsy. Clinical signs included hair loss on ventral abdomen or hind limbs, and clear or red material on body surfaces one hour after dosing seen in 92% of rats in the 320 mg/kg/day group and 33% of males and 75% of females in 80 mg/kg/day rats. The finding seen shortly after dosing did not persist to the next observation point. No clinical findings were observed in 20 mg/kg/day rats. Mean body weights in males were 13% and 8% lower and weight gain was 42% and 26% less than controls by the end of the exposure period in the 320 and 80 mg/kg/day groups, respectively. Changes in food consumption varied weekly, but were only statistically significantly decreased during the second week of exposure in the 320 mg/kg/day group males. No significant PEB Bottoms related effects on FOB parameters or locomotor activity were observed in males tested during study wk 5 or females on LD 4. No hematology findings were observed other than a decrease in mean absolute and/or % eosinophils in 80 mg/kg/day males and 320 mg/kg/day animals of both sexes. Serum chemistry parameters were unaffected by treatment at all dose levels. Increases of 10% in mean and 20-25% relative kidney weights in 80 and 320 mg/kg/day males and increased mean [20%] and relative liver weights [27-37%] in 320 mg/kg day rats of both sexes correlated with microscopic findings [mineralization, multifocal deposits and irregular basophilic material in male kidneys, and hepatocellular hypertrophy, respectively]. Increased thyroid gland weights of 10-15% compared to controls in both sexes and decreased thymus weights of 23% in females in the 320 mg/kg/day group correlated with microscopic changes [follicular cell hypertrophy in the thyroid, and thymic atrophy in females, respectively], but were not seen in the 80 and 20 mg/kg/day animals. The NOAEL for systemic toxicity was thus 20 mg/kg/day whereas the study LOAEL was 80 mg/kg/day based on decreased body weight and/or food consumption, and organ weight changes.