1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to GLP and guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

EC Number:

228-846-8

EC Name:

1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

Cas Number:

6362-80-7

Molecular formula:

C18H20

IUPAC Name:

(4-methyl-4-phenylpent-1-en-2-yl)benzene

Details on test material:

- Name of test material (as cited in study report): , 2,4-diphenyl-4-methyl-1-pentene [English name: 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene
- Analytical purity: 96.97%
- Impurities (identity and concentrations): 2,4-Diphenyl-4-methyl-2-pentene 2.54%)
- Storage condition of test material: Stored in a cabinet in the test substance storage room of the testing facility at room temperature (nominal value: 23°C, measured values 18.9 - 25.9°C) and protected from light.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan (Hino Breeding Center)
- Age at study initiation: 7 weeks of age (upon receipt)
- Weight at study initiation: was 237-271 g in males, 154-184 g in test group females and 168-179 g in recovery group females (upon receipt)
- Diet: Free access to pellet diet (CRF-1, Oriental Yeast Co., Ltd.)
- Water: Free access to tap water in water bottles
- Acclimation period: A five day quarantine period was followed by a 16 day acclimation period (17 days for the recovery group females

ENVIRONMENTAL CONDITIONS
- Temperature: Nominal temperature of 23°C (measured values: 22-24°C)
- Humidity: Nominal humidity of 55% (measured values: 40-60%)
- Air changes: 12 times/hour (filter-sterilized fresh air).
- Photoperiod: 12-hour light/dark cycle (illumination: 6:00 am to 6:00 pm)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of 2,4-diphenyl-4-methyl-1-pentene was weighed (electronic balance PM2500, Mettler-Toledo) and dissolved in corn oil to a concentration of 144 mg/mL. Dosing preparations at 36 and 9 mg/mL were prepared by serial dilution of the 144 mg/mL solution. Correction for the content of test substance was made at preparation


VEHICLE
- Amount of vehicle: 5 mg/mL
- Lot/batch no: Lot No. V4M3466 (expiration date: October 4, 2009), Lot No. V5F6057 (expiration date: May 29, 2010) and Lot No. V5P7775 (expiration date: October 25, 2010), Nacalai Tesque Inc

Details on mating procedure:

Males that were dosed for 14 days and test group females in the same groups were housed together on a one-to-one basis. The mating period was limited to 14 days, and the animals were housed together until mating was confirmed.

Confirmation of mating was done every morning at about the same time, and females with vaginal plugs or sperm in the vaginal smear were considered to be mated animals. That day was regarded as day 0 of pregnancy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance concentrations in the dosing preparations used on the starting day of administration to males and females and on the final day of administration to males were determined by gas chromatography (GC-14B, Shimadzu Corp.). The results showed that the test substance content was 91.6-100.7% of the nominal concentration, which was within the specified range (100±10%). Therefore, the concentration of each dosing preparation was acceptable

Duration of treatment / exposure:

The administration period was set based on the OECD Guideline for Testing of Chemicals for Combined Repeat Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (422). Administration to males was for 14 days before the mating period and 28 days thereafter, a total of 42 days. Administration to test group females was for 14 days before the mating period, during the mating period and gestation period, and until day 6 of lactation, a total of 44 to 56 days. Half the animals in each group of males were assigned to a 14-day recovery period following the 42-day administration period. For the recovery group females, a 14-day recovery period followed a 42-day administration period. The starting day of administration was day 1 of administration, and the day following the final administration was day 1 of recovery.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

45 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

180 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

720 mg/kg bw/day (nominal)

Details on study design:

- Dose selection rationale:
Dose levels were selected from the results of a preliminary combined repeat dose toxicity study with reproduction/developmental toxicity screening test by oral administration of 2,4-diphenyl-4-methyl-1-pentene to rats (dose levels: 0, 250, 500 and 1000 mg/kg, 5 males per group). In the 1000 mg/kg group, 1 animal died and decrease in body weight was observed. However, no abnormalities in clinical signs, body weight, food consumption or necropsy findings were observed in the 500 mg/kg group. Therefore, in this study an intermediate dose between 1000 mg/kg and 500 mg/kg of 720 mg/kg was selected as the high dose, and lower doses were set at 180 and 45 mg/kg in a common ratio of 4. The control group received the vehicle (corn oil) at the same volume.

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males were observed for clinical signs and mortality twice daily during the administration period, before and after administration, once daily during the recovery period, and once on the day of necropsy. Females were observed for clinical signs and mortality twice daily during the administration period, before and after administration, and once on the day of necropsy. Recovery group females were observed for clinical signs and mortality twice daily during the administration period, before and after administration, once daily during the recovery period, and once on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweight was measured twice a week

FOOD CONSUMPTION:
Food consumption was measured twice a week for 14 days before the start of the mating period and from after the end of the mating period

OTHER:
Mated females were allowed to deliver spontaneously, and observation for abnormalities in delivery and completion of delivery every day from day 21 of pregnancy until 10:00 in the morning on day 25 of pregnancy. If delivery was completed by 10:00 in the morning, that day was regarded as day 0 of lactation.

Dams were observed for the condition of lactation once a day from day 0 to day 4 of lactation

Oestrous cyclicity (parental animals):

The estrous cycle of test group females was observed once a day from the starting day of administration until the day before confirmation of mating. Estrus which continued for 2 days was counted as a single estrus

Litter observations:

Pups (F1)

Total Number of Offspring, Number of Stillbirths, Number of Newborn Pups on Day 0 of Lactation, Sex Ratio on Day 0 of Lactation, Delivery Index, Birth Index and Live Birth Index

Observation at Birth
The pups were observed at birth for the total number of offspring, sex, number of stillbirths, number of newborn pups and the presence of external abnormalities. Stillborn pups were necropsied, then fixed in 20 vol% neutral buffered formalin and preserved in 10 vol% neutral buffered formalin.

Observation of Pups
Pups were observed for general condition and mortality once a day. Pups that died were necropsied, then fixed in 20 vol% neutral buffered formalin and preserved in 10 vol% neutral buffered formalin.

Body Weight
Body weight was measured on day 0 (day of birth) and day 4 of lactation (electronic balance: PG2002-S, Mettler-Toledo).

Necropsy
The pups were sacrificed by exsanguination from the abdominal aorta under diethyl ether anesthesia on day 4 of lactation and necropsied.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
Males: The brain (cerebrum, cerebellum, medulla oblongata), pituitary, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, testes and epididymis were weighed (electronic balance: AB204, Mettler-Toledo). The relative weight of each organ was calculated by dividing its weight by the final body weight. The pituitary and thyroids were weighed after being fixed overnight in 20 vol% neutral buffered formalin. These organs and the lungs, trachea, pancreas, salivary glands (sublingual and submandibular), esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, lymph nodes (submandibular and mesenteric), urinary bladder, seminal vesicles, prostate, parathyroid, spinal cord, sciatic nerve, eyeballs, Harderian gland, sternum and femur were fixed in 20 vol% neutral buffered formalin. However, the testes and epididymis were fixed in Bouin’s solution for 2-3 hours, then re-fixed in 90 vol% ethanol, and eyeballs were fixed overnight in glutaraldehyde/formalin, then re-fixed in 20 vol% neutral buffered formalin.

Females: Were sacrificed by exsanguination and necropsied after determining the numbers of corpora lutea and implantations. In each group, the animals in which urinary, hematological and blood chemical examinations were not conducted were sacrificed by exsanguination from the abdominal aorta under anesthesia by intraperitoneal administration of sodium pentobarbital (40 mg/kg), and necropsied after determining the numbers of corpora lutea and implantations. The brain (cerebrum, cerebellum, medulla oblongata), pituitary, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, ovaries and uterus were weighed (electronic balance: AB204, Mettler-Toledo). The relative weight of each organ was calculated by dividing its weight by the final body weight. The pituitary and thyroids were weighed after being fixed overnight in 20 vol% neutral buffered formalin. These organs and the lungs, trachea, pancreas, salivary glands (sublingual and submandibular), esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, lymph nodes (submandibular and mesenteric), urinary bladder, vagina, parathyroid, spinal cord, sciatic nerve, eyeballs, Harderian gland, sternum, femur and mammary glands were fixed in 20 vol% neutral buffered formalin. However, the eyeballs were first fixed overnight in glutaraldehyde/formalin, then re-fixed in 20 vol% neutral buffered formalin.

The dam which did not deliver by 10:00 on day 25 of pregnancy was necropsied after exsanguination from the abdominal aorta under anesthesia by intraperitoneal administration of sodium pentobarbital (40 mg/kg), and pregnancy status was confirmed by the presence or absence of implantations. The organs and tissues listed above were fixed in 20 vol% neutral buffered formalin and preserved in 10 vol% neutral buffered formalin. However, the eyeballs were first fixed overnight in glutaraldehyde/formalin, then re-fixed in 20 vol% neutral buffered formalin.


HISTOPATHOLOGY: Yes
Males: For animals necropsied at the end of the administration period, paraffin-embedded specimens of the heart, lung, trachea, liver, pancreas, salivary glands (sublingual and submandibular), esophagus, stomach, duodenum, jejunum, ileum, cecum, rectum, colon, thymus, spleen, lymph nodes (submandibular and mesenteric), kidney, urinary bladder, testis, epididymis, seminal vesicle, prostate, pituitary, adrenal, thyroid, parathyroid, brain (cerebrum, cerebellum, medulla oblongata), spinal cord, sciatic nerve, eyeball, Harderian gland, bone marrow (sternum and femur) and bone (sternum and femur) were prepared. The excised organs and tissues were preserved in 10 vol% neutral buffered formalin. For the 720 mg/kg group and control group, HE-stained tissue specimens were prepared and examined histopathologically. PAS-hematoxylin-stained tissue specimens of testes were prepared. HE-stained tissue specimens of the liver and thyroid, which showed difference between the 720 mg/kg group and control group in the number of animals with abnormality, were also prepared for the 180 and 45 mg/kg groups and examined histopathologically. HE-stained specimens of the kidney, in which effects of test substance administration were suspected in the 720 mg/kg group, were also prepared for the 180 and 45 mg/kg group and examined histopathologically. PAS-hematoxylin-stained tissue specimens of the kidneys of all animals were also prepared and examined histopathologically.

Females: For 6 animals with the lowest animal numbers in each group and the animals that died in the 720 mg/kg group, paraffin-embedded specimens of the heart, lung, trachea, liver, pancreas, salivary glands (sublingual and submandibular), esophagus, stomach, duodenum, jejunum, ileum, cecum, rectum, colon, thymus, spleen, lymph nodes (submandibular and mesenteric), kidney, urinary bladder, ovary, uterus, vagina, pituitary, adrenal, thyroid, parathyroid, brain (cerebrum, cerebellum, medulla oblongata), spinal cord, sciatic nerve, eyeball, Harderian gland, bone marrow (sternum and femur), bone (sternum and femur) and mammary gland were prepared. The excised organs and tissues were preserved in 10 vol% neutral buffered formalin. Then for 6 animals with the lowest animal numbers in the 720 mg/kg group and control group and the animals that died in the 720 mg/kg group, HE-stained tissue specimens were prepared and examined histopathologically. HE-stained tissue specimens of the liver, kidney and thyroid, which showed difference between the 720 mg/kg group and control group in the number of animals with abnormality, were also prepared for 6 animals with the lowest animal numbers in 180 and 45 mg/kg groups and examined histopathologically. PAS-hematoxylin-stained tissue specimens of the kidneys of 6 animals with the lowest animal numbers in each group were also prepared and examined histopathologically

Statistics:

Body weight of pups was calculated as the mean and total litter values, in the unit of mean per dam
Estrus counts, number of days required for mating, gestation period [day of delivery (day 0 of lactation) – day of mating confirmation], number of corpora lutea, number of implantations, implantation index [(number of implantations / number of corpora lutea) ×100], total number of offspring (number of newborn pups + number of stillbirths), number of newborn pups, number of stillbirths, delivery index [(total number of offspring / number of implantations) ×100], birth index [(number of newborn pups on day 0 of lactation / number of implantations) ×100], live birth index [(number of newborn pups on day 0 of lactation / total number of offspring) ×100], number of live pups on day 4 of lactation, viability index on day 4 of lactation [(number of live pups on day 4 / number of newborn pups on day 1 of lactation) ×100], external abnormalities [(number of pups with external abnormalities / number of newborn pups) ×100] and sex ratio (males/females). Equality of variance was tested by Bartlett’s test, and Dunnett’s test was used if equality of variance was observed. However, if equality of variance was not observed, a Duunett-type rank test was used.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Number of Estrus Cases:
The number of estrus cases during the pre-mating administration period (14 days) in the 720 mg/kg group tended to be low, although it showed no significant difference from the control group. The number of estrus cases during the pre-mating administration period (14 days) in the 180 and 45 mg/kg groups showed no significant difference from the control group.

Number of Conceiving Days, Copulation Index, Number of Pregnant Females and Fertility Index:
All animals in each group copulated. The copulation index in each group was 100.0%. There was no significant difference in the number of conceiving days between any test administration group and the control group.

There was 1 non-pregnant in both the 720 and 180 mg/kg groups. The fertility index showed no significant difference between any test administration group and the control group.

Gestation Period:
The gestation period showed no significant difference between any test administration group and the control group.

Number of Corpora Lutea, Number of Implantations and Implantation Index:
In the 720 mg/kg group, the numbers of corpora lutea and implantations were significantly low compared to the control group, and the implantation index tended to be low, though without significant difference. In the 180 and 45 mg/kg groups, there was no significant difference from the control group in the numbers of corpora lutea and implantations or the implantation index.

Gestation Index, Delivery Condition and Lactation Condition:
The gestation index was 100.0% in each group.

No abnormalities in delivery condition were observed in any group.

In nursing condition, faulty nest-building and faulty nipple development were observed in 1 dam in the 180 mg/kg group (No. F03353), but they judged to be incidental because the changes lacked dose relation.

Effect levels (P0)

1

Dose descriptor:

NOEL

Remarks:

reproductive-developmental toxicity

Effect level:

180 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on effects on testis weight observed at 720 mg/kg

2

Dose descriptor:

NOEL

Remarks:

reproductive-developmental toxicity

Effect level:

180 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on effects on estrous cycle, number of copora lutea, number of implanations and implantation index observed at 720 mg/kg/day

Results: F1 generation

Details on results (F1)

Total Number of Offspring, Number of Stillbirths, Number of Newborn Pups on Day 0 of Lactation, Sex Ratio on Day 0 of Lactation, Delivery Index, Birth Index and Live Birth Index:
In the 720 mg/kg group, the total number of offspring and number of newborn pups on day 0 of lactation were significantly low compared to the control group, and the delivery index and birth index tended to be low, although without significant difference. In the 180 and 45 mg/kg groups, there were no significant differences from the control group in total number of offspring, number of stillbirths, number of newborn pups on day 0 of lactation, sex ratio on day 0 of lactation, delivery index, birth index or live birth index.

Clinical Signs of Pups, Number of Live Pups on Day 4 of Lactation, Sex Ratio on Day 4 of Lactation, Survival Rate on Day 4 of Lactation and External Abnormalities:
In the 720 mg/kg group, the number of live pups on day 4 of lactation was significantly lower than the control group, although there was no significance in the survival rate on day 4 of lactation. In the 180 and 45 mg/kg groups, there were no significant differences from the control group in the number of live pups on day 4 of lactation, sex ratio on day 4 of lactation or survival rate on day 4 of lactation.

No external abnormalities in live pups were observed in any group.

In clinical signs of pups, hypothermia was observed in 1 litter in the 180 mg/kg group (No. F03353, the dam in which faulty nest-building and faulty nipple development were observed), but it was judged to be an incidental change.

Body Weight of Pups:
In the 720 mg/kg group, the mean body weight of males on days 0 and 4 of lactation was significantly higher than the control group, although without significant difference, the mean body weight of females on days 0 and 4 of lactation tended to be higher, mean litter weight was significantly high on days 0 and 4 of lactation, and total litter weight was significantly low on days 0 and 4 of lactation. In the 180 and 45 mg/kg groups, there were no significant differences from the control group in mean body weight of males or females on day 0 and 4 of lactation, mean litter on days 0 and 4 of lactation, or total litter weight on days 0 and 4 of lactation.

Necropsy of Dead Pups:
No abnormalities were observed in any group.

Necropsy of Surviving Pups:
No abnormalities were observed in any group.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

180 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on effects on the total number of offspring, newborn on day 0 of lactation, delivery index, birth index and number of live pups on day 4 of lactation observed at 720 mg/kg.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The no-effect level for reproductive-developmental toxicity was 180 mg/kg/day for males because effects on testis weight were observed at 720 mg/kg, and was 180 mg/kg/day for females because effects on estrous cycle, number of corpora lutea, number of implantations and implantation index were observed at 720 mg/kg. The no-effect level for pups was 180 mg/kg/day because effects on the total number of offspring, newborn on day 0 of lactation, delivery index, birth index, and number of live pups on day 4 of lactation were observed at 720 mg/kg.