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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

28 day repeat dose oral toxicity study combined with a reproductive/ developmental toxicity screening test:

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.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

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)

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.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

45 mg/kg bw/day (nominal)

Dose / conc.:

180 mg/kg bw/day (nominal)

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.

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.

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.

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

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

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.

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.

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

720 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Prenatal development toxicity study (OECD 414:)

Summary:

The purpose of this study was the assessment of the influence of the test item, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague-Dawley rat. Three groups of 20 females received test item at doses of 60, 200 or 600 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Clinical observations, body weight, food consumption and water consumption were recorded.

Adult females were examined macroscopically at necropsy on Day 20 after mating, the gravid uterus weight was recorded and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results:

One animal receiving 600 mg/kg/day was found dead on the morning of Day 7. The cause of death was undetermined, but was considered un-related to treatment.

Two animals receiving 600 mg/kg/day showed evidence of blood loss from the vagina, at the end of gestation; the litter of one of these females contained a high number of resorbed implantations.

 

At 600 mg/kg/day, initial body weight gain (Days 6-9) was markedly low and overall body weight gain was low, and correlated with low food and low water intake at this dose. Adjusted body weight gain (adjusted for gravid uterine weight) at 600 mg/kg/day was 5 g, compared to 31 g of Control.

Initial body weight gain (Days 6-9) was markedly low at 200 mg/kg/day and was low at 60 mg/kg/day. Overall gains were unaffected at both doses; however, adjusted weight gain was low at 200 mg/kg/day.

 

There was no effect of treatment on gravid uterine weight and adults and fetuses were macroscopically normal.

The number of corpora lutea, implantations, sex ratio of males to females and placental and fetal weights were unaffected by treatment.

Mean post-implantation loss was high at 600 mg/kg/day and resulted in less fetuses at this dose.

The higher incidences of delayed ossification of some fetal bones at 600 mg/kg/day were considered to represent a transient stage in development and not to be detrimental to the growth, development and survival of the fetuses.

Conclusion:

Based on the results of this study, the maternal and embryo-fetal No-Observed-Adverse- Effect-Level (NOAEL) was 200 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

Prenatal development toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 23 November 2016; Experimental completion date : 16 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Animals:
Strain/Species: Crl:CD(SD) rat.
Supplier: Charles River (UK) Ltd.
Number of animals ordered:
90 females.
Spare animals (additional females ordered for possible study use) were removed from the study room after treatment commenced.
Stock males were removed from the study room before treatment commenced.
Duration of acclimatization: Five days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation): Nominally 71 days old.
Weight range of the animals at the start of the study (Day 0 of gestation): 237 to 301 g.

Environmental control:
Rodent facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%.
Lighting: Artificial lighting, 12 hours light ; 12 hours dark

Animal accomodation:
Cages:
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.

Cage distribution: The cages constituting each group were blocked by group and mounted in batteries.

Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage:
Acclimatization: up to four
During pairing: one (stock) male and one female
Gestation: one female

Environmental enrichment:
Aspen chew block, plastic shelter.

Diet Supply:
Diet: SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water Supply:
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

TEST ITEM PREPARATION:
Formulation:
Group 1: Vehicle (Dose:0 mg/kg/day; Nominal concentration: 0 mg/mL; Formulated concentration: 0 mg/mL; Volume dose: 5 mL/kg)
Group 2: Test item (Dose:60 mg/kg/day; Nominal concentration: 12 mg/mL; Formulated concentration: 12.72 mg/mL; Volume dose: 5 mL/kg)
Group 3: Test item (Dose:200 mg/kg/day; Nominal concentration: 40 mg/mL; Formulated concentration: 42.4 mg/mL; Volume dose: 5 mL/kg)
Group 4: Test iem (Dose:600 mg/kg/day; Nominal concentration: 120 mg/mL; Formulated concentration: 127.2 mg/mL; Volume dose: 5 mL/kg)

Correction factor:
A correction factor of 1.06 was used for purity of the test item, when calculating quantities of test item used during dose preparation.

Method of Preparation:
Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the required amount of test item. This was magnetically stirred until all of the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume. The formulation was then mixed using a high shear homogenizer until homogenous.
The remaining concentrations were made using the same method in ascending order.

Frequency of preparation:
Weekly. All formulations were administered within the known stability period.

Storage of formulation:
Refrigerated (2 to 8°C).

Test item accounting:
Detailed records of test item usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

DOSE ADMINISTRATION:
The study consisted of one control and three treated groups identified as follows:

Group 1: Control (vehicle)
Dose: 0 mg/kg/day
Number of animals: 20 Female

Group 2: Test item
Dose: 60 mg/kg/day
Number of animals: 20 Female

Group 3: Test item
Dose: 200 mg/kg/day
Number of animals: 20 Female

Group 4: Test item
Dose: 600 mg/kg/day
Number of animals: 20 Female

Administration:
Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg.
Volume dose: 5 mL/kg bodyweight
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Formulation: A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Formulations from 2 mg/mL to 200 mg/mL have been shown to be stable in corn oil for up to seven days when stored refrigerated (nominally +4°C) and six hours at ambient temperature (15-25°C), when protected from light.

Achieved concentration: Samples of the first and last formulations that were prepared for treatment were analyzed for achieved concentration of the test item.

Method of analysis: High performance liquid chromatography (HPLC)

Results:
The mean concentrations were within 4% of the nominal concentration, confirming the accuracy of formulation. Percentage differences from mean values were within 1% confirming the continued accuracy of the analytical procedure.

Procedural recovery values remained within the validated limits, confirming the continued accuracy of the method.

Details on mating procedure:

Males: Stock animals supplied by Charles River (UK) Ltd.
Male/female ratio 1:1
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation: When positve evidence of mating was detected.

A colony of stud males, from the same source as the females, was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating.

Frequency of treatment:

Once daily at approximately the same time each day.

Duration of test:

20 days:
Day 0 - 6: Mating
Day 6 - 19: Treatment
Day 20: Necropsy

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

20 females per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for this study (0, 60, 200 and 600 mg/kg/day) were selected based on the effects of a preliminary embryo-fetal study that investigated dose levels of 180, 360 and 720 mg/kg/day. In that study there were no unscheduled deaths and no signs associated with dosing; however at the end of gestation, one animal treated at 720 mg/kg/day had piloerection, hairloss on fore/hindlimbs and ventral body surface and encrustations on the forelimbs and low overall body weight gain (33g vs group mean of 82g) that was considered an individual animal response to treatment. At 180, 360 or 720 mg/kg/day, overall body weight gain was low with relationship to treatment and food consumption was marginally low. Water consumption was high at 720 mg/kg/day. At 360 or 720 mg/kg/day, gravid uterine weight was low and the number of total resorptions and pre- and post-implantation losses was high in some litters.

Dose levels of 60, 200 and 600 mg/kg/day were selected for this study. A dose of 600 mg/kg/day was expected to result in some maternal toxicity but hopefully reduce the level of post-implantation loss, therefore protecting the number of live fetuses available for detailed fetal examination.

Maternal examinations:

MORTALITY:
A viability check was performed near the start and end of each working day. A complete necropsy was performed in all cases of mortality.

CLINICAL OBSERVATIONS:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

SIGNS ASSOCIATED WITH DOSING:
Detailed observations were recorded daily at the following times in relation to dose administration:
-One to two hours after completion of dosing
-As late as possible in the working day

CLINICAL SIGNS:
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT:
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION:
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

WATER CONSUMPTION:
Water consumption was recorded by weight for each animal, daily, from Days 0-19 inclusive, after mating.

TERMINAL INVESTIGATIONS:
METHOD OF KILL:
Method of kill for all adult animals: Carbon dioxide asphyxiation.
Method of kill for fetuses: Chilling on a cool plate (approximately 0°C)

NECROPSY:
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule: Animals surviving at the end of the scheduled study period were killed on Day 20 after mating.
Sequence: To allow satisfactory inter-group comparison

Ovaries and uterine content:

For females surviving to term, the following was recorded:

Uterus: Gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals (including those prematurely sacrificed, where possible):

For each ovary/uterine horn, the number of:
- Corpora lutea
- Implantation sites
- Resorption sites (classified as early or late).
- Fetuses (live and dead).

Apparently non-pregnant animals: The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique].

Fetal examinations:

FETAL EXAMINATION AND PROCESSING:
Examination of all viable fetuses and placentae:
Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.

Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.

Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning. IMS fixed fetuses were processed and stained with Alizarin Red.

FETAL PATHOLOGY EXAMINATION:
Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses: Assessed for skeletal development and abnormalities.

FETAL, LITTER and PLACENTAL WEIGHTS:
Mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights.
Mean placental weight was also calculated for each litter.

Group mean values and SD were calculated using individual litter mean values.

DETAILED FETAL EXAMINATION:
Observations on fetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants.
Findings observed were classified, according to severity and incidence, as:
Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect.
Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs and thoracolumbar vertebra.

Statistics:

The following data types were analyzed at each timepoint separately:

- Body weight, using absolute weights and gains over appropriate study periods
- Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
- Water consumption, over appropriate study periods
- Litter size and survival indices
- Fetal, placental and litter weight

The following comparisons were performed:
Group 1 vs 2, 3 and 4

Indices:

Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) / Number of corpora lutea x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = (Number of implantations - Number of live fetuses) / Number of implantations x 100

All group values and SD (as appropriate) were calculated from the individual litter values.

Historical control data:

The Sprague-Dawley Crl:CD(SD) strain was used because of the historical control data available at the laboratory (see attached background material)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One animal treated at 600 mg/kg/day had red discharge from the vagina on Days 17 and 18 of gestation and had piloerection and was pale on Days 17-20 and another female at the same dose had aqueous, brown discharge from the vagina on Day 20.

At 600 mg/kg/day, one animal had salivation at 1-2 hours after dosing on Day 19 of gestation.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal (No. 63) was found dead on Day 7 after mating, following a single dose at 600 mg/kg/day on Day 6. The animal did not show any clinical signs or evidence of toxicity following one administration. Macroscopic examination revealed distension of the stomach with food material.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall body weight gain (Days 6-20 of gestation) at 600 mg/kg/day was low, when compared with Control (75%) and adjusted gain (excluding gravid uterine weight) was 16% of Control. Overall body weight gain at 200 mg/kg/day was comparable with Control, but adjusted gain was low (74% of Control). Overall body weight and adjusted gains were unaffected by treatment at 60 mg/kg/day.

Following commencement of treatment, initial bodyweight gain (Days 6-9 of gestation) at 60 mg/kg/day was low (69% of Control) and was markedly low at 200 or 600 mg/kg/day (46 or 15% of Control, respectively). Thereafter, bodyweight gain to Days 9-20 was similar to Control at 60 or 200 mg/kg/day, but remained low (82%) at 600 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption during Days 6-9 of gestation was slightly low in animals treated at 200 or 600 mg/kg/day (87 or 74%, respectively) and was marginally low during Days 10-19 of gestation at 600 mg/kg/day (88%).

Description (incidence and severity):

See food consumption.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

When compared with Control, overall water consumption (Days 6-19 of gestation) in animals treated at 600 mg/kg/day, was high (136%).

Overall water intake was unaffected by treatment at 60 or 200 mg/kg/day.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings in the adults, considered to be related with treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

See details on maternal toxic effects.
At 600 mg/kg/day, the number of post implantation losses was high and resulted in a lower number of live fetuses at Day 20 of gestation, when compared with Control

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

See summary data for live offspring.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

See details on maternal toxic effects and summary data.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Included in early/late resorptions.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

1 animal at 60 mg/kg/day was non-pregnant. Reason for non-pregnancy not specified but not considered related to treatment and no dose-response relationship, as all animals pregnant at higher doses.

Details on maternal toxic effects:

LITTER RESPONSES:
One animal treated at 60 mg/kg/day was not pregnant and one animal at 600 mg/kg/day was found dead on Day 7; therefore there were 20, 19, 20 and 19 females at 0, 60, 200 or 600 mg/kg/day respectively, with live fetuses at necropsy for evaluation.

LITTER DATA:
At 600 mg/kg/day, the number of post implantation losses was high and resulted in a lower number of live fetuses at Day 20 of gestation, when compared with Control and was also just outside the upper limit of the historical control data (HCD) range; the number of corpora lutea and implantations and the ratio of males to females were unaffected by treatment.

The number of corpora lutea, implantations, resorptions (early or late), implantation losses (pre- and post) and live young and the ratio of males to females at 60 or 200 mg/kg/day were unaffected by treatment.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Abnormalities:

effects observed, treatment-related

Localisation:

other: post-implantation losses

Description (incidence and severity):

Increase at 600 mg/kg/day compared to control

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal and placental weights unaffected by treatment.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

At 600 mg/kg/day, the number of post implantation losses was high and resulted in a lower number of live fetuses at Day 20 of gestation, when compared with Control.
See summary of results form mean number and percent of live offspring.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Ratio of males to females was unaffected by treatment.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

When compared with Control, mean litter weight at 600 mg/kg/day was marginally, but not statistically significantly low (92%). This was considered a result of the marginally smaller litter size.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to summary of Fetal examinations.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to summary of Fetal examinations.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to summary of Fetal examinations.

Details on embryotoxic / teratogenic effects:

LITTER DATA:
At 600 mg/kg/day, the number of post implantation losses was high and resulted in a lower number of live fetuses at Day 20 of gestation, when compared with Control and was also just outside the upper limit of the historical control data (HCD) range; the number of corpora lutea and implantations and the ratio of males to females were unaffected by treatment.

The number of corpora lutea, implantations, resorptions (early or late), implantation losses (pre- and post) and live young and the ratio of males to females at 60 or 200 mg/kg/day were unaffected by treatment.

PLACENTAL, LITTER and FETAL WEIGHTS:
When compared with Control, mean litter weight at 600 mg/kg/day was marginally, but not statistically significantly low (92%). This was considered a result of the marginally smaller litter size. Fetal and placental weights were unaffected by treatment.

Mean placenta, litter and fetal weights were unaffected by maternal treatment at 60 or 200 mg/kg/day.

FETAL PATHOLOGY:
There were no major fetal abnormalities related with treatment.

When compared with Control, there were higher incidences of delayed ossification at 600 mg/kg/day (cranial centres and thoracic vertebrae) and dilated ureter that were within the Historical Control Data range (HCD). The slight increase of fetuses at 600 mg/kg/day, with delayed ossification of sternebrae and pelvic bones, that marginally exceeded the HCD range, were considered to be a transient stage in development and not to be detrimental to growth, development or survival.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

skeletal malformations

Remarks on result:

other:

Abnormalities:

effects observed, non-treatment-related

Localisation:

other: Skeletal: cranial centres, thoracic vertebrae, sternbrae, pelvic bones, dilated ureter

Description (incidence and severity):

examination of the fetuses at 600 mg/kg/day revealed higher incidences of delayed ossification of some bones and there were a higher number of fetuses with dilated ureter compared with Controls.
Some findings slightly exceeded the Historical Control Data range, but were considered to represent a transient stage in development and none were considered to be detrimental to fetal growth, development or survival.

Developmental effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Full results tables (for group data) and appendixes (for individual animal data) are attached as background material.

Data summarised below:

Number of pregnant and non-pregnant dams:

Dose (mg/kg/day)	Number of pregnant dams	Number of non-pregnant dams
0	20	0
60	19*	1
200	20	0
600	20**	0

*Reason for non-pregnancy not specified

** 1 animal died in this group but was pregnant

Number of dams with abortions, early-deliveries, stillbirths, resorptions, and/or dead foetuses: (For full data see Table 6 and Appendix 8 in attached background material).

Dose (mg/kg/day)	0	60	200	600
Abortions	0	0	0	0
Early deliveries	0	0	0	0
Still births	0	0	0	0
Resorptions*	0.7	0.7	0.9	1.5

*resoprtion values based on group mean values for total resorptions (early and late), and also including for dead foetuses.

Pre- and post-implantation loss, number and percent: (For full data see Table 6 and Appendix 8 in attached background material).

Dose (mg/kg/bw)	0	60	200	600
Number pre-implantation loss**	349 – 318 = 31	317 – 297 = 20	352 – 334 = 18	325 – 323 = 2
% pre-implantation loss*	8.8%	7.0%	4.9%	3.8%
Number post-implantation loss***	318 – 305 = 13	297 – 284 = 13	334 – 317 = 17	323 – 273 = 50
% post implantation loss*	4.1%	4.9%	5.0%	10.1%

*% values based on group mean figures.

**calculated as group total corpora lutea - group total implantations

***calculated as group total implantations - total number of live foetuses.

Body weight, body weight change and gravid uterine weight, including optionally, body weight change corrected for gravid uterine weight: (For full data see Table 3 and Appendix 3 and 4 in attached backgroun material).

Group mean values (g):

Dose (mg/kg/day)	Body weight on Day 6	Terminal Body weight on Day 20	Body weight change Days 6-20	Gravid Uterine Weight	Adjusted Body weight Day 20	Adjusted body weight change Days 6-20
0	292	414	122	91	323	31
60	296	421	125	90	331	35
200	298	415	117	94	320	23
600	292	383	91	86	298	5

 

Mean number and percent of live offspring: (For full data see Table 6 and Appendix 8 in attached background material).

Dose (mg/kg/bw)	0	60	200	600
Mean number live offspring*	15.3	14.9	15.9	14.4
% live offspring**	95.9	95.1	95	89.9

*based on group mean values

** calculated as 100% - % post-implantation loss

Mean foeatal/pup bodyweight by sex and sexes combined: (For full data see Table 7 and Appendix 9 in attached background material).

group mean values (g):

Dose (mg/kg/day)	Placental Weight	Litter Weight	Litter Size	Male Fetal Weight	Female Fetal Weight	Overall Fetal Weight
0	0.53	56.75	15.25	3.84	3.62	3.73
60	0.55	55.73	14.95	3.84	3.63	3.73
200	0.54	58.12	15.85	3.77	3.57	3.67
600	0.56	52.30	14.37	3.70	3.5	3.60

Number and percent of foetuses and litters with malformations (including runts) and/or variation as well as description and incidences of malformations and main variations (and/or retardation)

For full data see Tables 8, 9, 10 and Appendix 10 in attached background material).

Fetal examinations – major abnormality findings -group incidences

Dose (mg/kg/bw)	0	60	200	600
Number of foetuses with malformation	3 out of 305 examined	1 out of 284 examined	0 out of 317 examined	3 out of 273 examined
% of foetuses with malformation	0.98%	0.35%	0%	1.1%
Number of litters with malformation	3 out of 20 examined	1 out of 19 examined	0 out of 20 examined	3 out of 19 examined
% of litters with malformation	15%	5%	0%	15.8%

 

Fetal examinations – minor visceral abnormality and necropsy findings – group incidences

Dose (mg/kg/bw)	0	60	200	600
Number of foetuses with malformation	20 out of 152 examined	19 out of 141 examined	24 out of 157 examined	34 out of 136 examined
% of foetuses with malformation	13.1%	13.5%	15.3%	25%
Number of litters with malformation	12 out of 20	12 out of 19	13 out of 20	15 out of 19
% of litters with malformation	60%	63%	65%	79%

Fetal examinations - minor skeletal abnormality and variants findings - group incidences

Dose (mg/kg/bw)	0	60	200	600
Number of foetuses with malformation	118 of 160 examined	105 of 143 examined	119 of 160 examined	119 of 137 examined
% of foetuses with malformation	73.8%	73.4%	74.4%	86.9%

Conclusions:

Based on the results of this study, the maternal and embryo-fetal No-Observed-Adverse- Effect-Level (NOAEL) was 200 mg/kg/day.

Executive summary:

Summary:

The purpose of this study was the assessment of the influence of the test item, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague-Dawley rat. Three groups of 20 females received test item at doses of 60, 200 or 600 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Clinical observations, body weight, food consumption and water consumption were recorded.

Adult females were examined macroscopically at necropsy on Day 20 after mating, the gravid uterus weight was recorded and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results:

One animal receiving 600 mg/kg/day was found dead on the morning of Day 7. The cause of death was undetermined, but was considered un-related to treatment.

Two animals receiving 600 mg/kg/day showed evidence of blood loss from the vagina, at the end of gestation; the litter of one of these females contained a high number of resorbed implantations.

 

At 600 mg/kg/day, initial body weight gain (Days 6-9) was markedly low and overall body weight gain was low, and correlated with low food and low water intake at this dose. Adjusted body weight gain (adjusted for gravid uterine weight) at 600 mg/kg/day was 5 g, compared to 31 g of Control.

Initial body weight gain (Days 6-9) was markedly low at 200 mg/kg/day and was low at 60 mg/kg/day. Overall gains were unaffected at both doses; however, adjusted weight gain was low at 200 mg/kg/day.

 

There was no effect of treatment on gravid uterine weight and adults and fetuses were macroscopically normal.

The number of corpora lutea, implantations, sex ratio of males to females and placental and fetal weights were unaffected by treatment.

Mean post-implantation loss was high at 600 mg/kg/day and resulted in less fetuses at this dose.

The higher incidences of delayed ossification of some fetal bones at 600 mg/kg/day were considered to represent a transient stage in development and not to be detrimental to the growth, development and survival of the fetuses.

Conclusion:

Based on the results of this study, the maternal and embryo-fetal No-Observed-Adverse- Effect-Level (NOAEL) was 200 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

28 day repeat dose oral toxicity study combined with a reproductive/ developmental toxicity screening test:

The no-effect level for reproduction/ developmental toxicity was considered to be 180 mg/kg/day. An expert review of the study results, attached, indicates classification as a reproductive toxicant is not necessary. Based on the results of a combined repeat dose study with the evaluation of reproduction/developmental toxicity (Test Guideline OECD 422), it is possible to suggest that classification of this chemical for effects upon reproduction/development are not justified. This opinion is based upon the following reasons:

1. A potential effect upon oestrous cyclicity prior to mating at the highest dose level was likely to be a chance event rather than a true effect of treatment. Female mating performance and fertility were unaffected by treatment.

2. The effect upon implantation number, corpora lutea and live litter size at the highest dose level may well reflect an adaptive change. This is because these effects were only seen at a dose level that was toxic to the adult animal. At lower dose levels there were no treatment effects upon reproduction.

3. The reduction in live litter size at the highest dose level was not seen with any effect upon offspring body weight gain from birth to Day 4 post partum. This indicates a lack of effect upon neonatal development.

Prenatal developmental toxicity study:

The effects observed in the OECD 414 study are not considered to be sufficient to justify classification.

Post-implantation loss was high at 600 mg/kg/day when compared to control values (as below), but not considered substantially high enough to justify classification.

Control (group mean): 4.1%

600 mg/kg/day (group mean): 10.1%

The group mean for the 6000 mg/kg/day is also only just outside the historical control data ranges for post-implantation losses: mean 5.61%, lower limit 2.7%, upper limit 9.8%.

The initial meternal toxicity at 600 mg/kg/day occurred concurrently with the observed high number of post implantation losses. The general condition of a small number of pregnant animals receiving 600 mg/kg/day deterioated at the end of gestation.

The observed post-implantation loss is therefore not considered conclusive evidence to justify classification.

Pathological examination of the fetuses at 600 mg/kg/day revealed higher incidences of delayed ossification of some bones (cranial centres, thoracic vertebrae, sternebrae and pelvic bones) and there were a higher number of fetuses with dilated ureter compared with Controls. Some findings slightly exceeded the Historical Control Data range, but were considered to represent a transient stage in development and none were considered to be detrimental to fetal growth, development or survival. These effects do therefore not justify classification.