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EC number: 202-896-0 | CAS number: 100-86-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
Toxicity to Reproduction (Screening for reproductive/developmental toxicity): This study is waived as a series of pre-natal developmental toxicity studies (read-across to PEA; CAS No. 60-12-8) in rats are available.
Effect on fertility: via oral route
- Endpoint conclusion:
- no study available
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Toxicity to Reproduction (Screening for reproductive/developmental toxicity): This study is waived as a series of pre-natal developmental toxicity studies (read-across to PEA; CAS No. 60-12-8) in rats are available.
Effects on developmental toxicity
Description of key information
Read-across to PEA (CAS No. 60-12-8) - Pre-natal developmental toxicity (dermal): Maternal NOAEL = 430 mg/kg bw/day; Developmental NOAEL = 140 mg/kg bw/day; NOAEL for viability and growth = 430 mg/kg bw/day (Equivalent or similar to OECD 414/GLP)
Read-across to PEA (CAS No. 60-12-8) - Pre-natal developmental toxicity (oral): Maternal and developmental NOAEL (oral: diet) were 266 mg/kg bw/day (Equivalent or similar to OECD 414/GLP)
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Please see the read-across justification.
- Reason / purpose for cross-reference:
- read-across source
- Dose descriptor:
- NOAEL
- Effect level:
- 430 mg/kg bw/day (nominal)
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- Abnormalities:
- no effects observed
- Dose descriptor:
- NOAEL
- Remarks:
- Developmental (DG21)
- Effect level:
- 140 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- skeletal malformations
- Dose descriptor:
- NOAEL
- Remarks:
- Viability and growth on DL21
- Effect level:
- 430 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- reduction in number of live offspring
- skeletal malformations
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- skeletal: rib
- Description (incidence and severity):
- All apparent delays in ossification and increased incidences of a cervical rib at the 7th cervical vertebrae that were observed in the Caesarean delivered fetuses in the 430 mg/kg bw/day dosage group were resolved by DL 21.
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 140 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- no
- Conclusions:
- In a Combined Dermal Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PEA (phenethyl alcohol) in Rats, the maternal NOAEL is 430 mg/kg/day. The 1400 mg/kg/day dosage caused mortality and reductions in body weight and feed consumption. The developmental NOAEL for pups Caesarean-delivered on day 21 of gestation is 140 mg/kg/day. Maternal dosages of 430 and 1400 mg/kg/day caused reductions in fetal weight with corresponding delays in fetal skeletal ossification and an increase in the incidence of cervical ribs. All 12 fetuses in the two litters that had live fetuses in the 1400 mg/kg/day dosage group had one or more gross, soft tissue and/or skeletal alterations. The 1400 mg/kg/day dosage also produced embryo/fetal lethality. The NOAEL for viability and growth in the offspring naturally delivered and euthanized on day 21 of lactation is 430 mg/kg/day. The maternal dosage of 1400 mg/kg/day increased the incidence of perinatal mortality. As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, several of the 24 live pups in the two litters that had live pups in the 1400 mg/kg/day dosage group on postpartum day 21 had a skeletal alteration. However, all apparent delays in ossification and increased incidences of a cervical rib at the 7th cervical vertebrae that were observed in the Caesarean delivered fetuses in the 430 mg/kg/day dosage group were resolved by DL 21.
- Executive summary:
In a combined dermal developmental and perinatal/postnatal reproduction toxicity study (58462) PEA (99.42%) or deionized water was administered dermally (occlusive) to 40 female Crl:CD(SD) rats at dose levels of 0, 140, 430 and 1400 mg/kg bw/day beginning on day 7 of presumed gestation and continuing through day 20 of presumed gestation. Twenty rats per dosage group were Caesarean-sectioned on day 21 of presumed gestation and 20 rats per dosage group were allowed to deliver their litters and were sacrificed on day 21 postpartum. Skin irritation was assessed using the Draize method.
In the rats assigned to caesarean-sectioning, mortality was significantly increased at 1400 mg/kg bw/day, in comparison to the vehicle control group value. Five and three rats in the 1400 mg/kg bw/day dosage group were found dead or humanely euthanized, respectively. Most of these rats had decreased motor activity, impaired or lost righting reflex, ataxia, hunched posture, mild or moderate dehydration (based on skin turgor), red perivaginal substance and vocalization before death. In addition, observations consistent with primary irritancy (erythema and flaking) occurred in most of the female rats that died or were euthanized. These skin reactions and clinical signs are consistent with observations noted in rats that survived to scheduled sacrifice; therefore, the deaths were attributed to treatment with Phenethyl Alcohol.
Observations consistent with primary irritancy occurred in female rats percutaneously administered Phenethyl Alcohol during the gestation period. These skin reactions included: flaking grade 2 (≥430 mg/kg bw/day) and erythema grades 1 through 3 and flaking grade 3 (1400 mg/kg bw/day). Each of these skin reactions, with the exception of erythema grade 3, occurred in significantly more rats in the 430 and/or 1400 mg/kg bw/day dosage groups, in comparison to the vehicle control group values. The onset and severity of the skin reactions was, in general, dependent on the dosage of Phenethyl Alcohol.
Clinical signs attributed to treatment with Phenethyl Alcohol included: decreased motor activity, impaired righting reflex, lost righting reflex, ataxia, hunched posture, ptosis, hyperreactivity to touch and/or in the nesting box, vocalization,mild, moderate or severe dehydration (based on skin turgor), cold extremities, moderate excess salivation, chromorhinorrhea, chromodacryorrhea, urine-stained abdominal fur, ungroomed coat, scab forming an abrasion on the back, clear and/or red perivaginal substance and scant feces. The number of affected rats at 1400 mg/kg bw/day was significantly increased in comparison to the vehicle control group values.
A statistically significant loss in body weight, coupled with reduced or significantly reduced feed consumption values, occurred at 1400 mg/kg bw/day for the entire dosage period, as compared to the vehicle control group values. Absolute and relative feed consumption values at 1400 mg/kg/day were 78% and 94% of the vehicle control group value, respectively, for DGs 7 to 21. Overall, body weight gains and absolute feed consumption values were significantly reduced for the entire gestation period, as compared to the vehicle control group values. At 1400 mg/kg bw/day, the average maternal body weight was significantly reduced beginning on DG 8 and continuing until DG 21.
Pregnancy occurred in 16 to 20 rats in each dosage group. As a result of the increased mortality that occurred in the 1400 mg/kg bw/day dosage group, Caesarean-sectioning observations on DG 21 were based on 20, 18, 20 and 9 pregnant rats in the 0 (Vehicle Control), 140, 430 and 1400 mg/kg bw/day dosage groups, respectively. At 1400 mg/kg bw/day, postimplantation loss (i.e., early and late resorptions and the percentage of resorbed conceptuses per litter) was increased or significantly increased, in comparison to the vehicle control group values. Seven of the nine rats in this dosage group had total litter loss (i.e., 100% resorbed conceptuses). These increases in postimplantation loss resulted in an overall significant reduction in the averages for litter size and live fetuses at 1400 mg/kg bw/day, in comparison to the vehicle control group values (1.3 per litter vs. 14.6 per litter in vehicle controls). At ≥430 mg/kg bw/day, the average fetal body weight (total, male and female) was significantly lower than the concurrent vehicle control group values. These values were also below the range observed historically at the Testing Facility. The combined fetal weight at 430 and 1400 mg/kg bw/day was 91% and 48% of the vehicle control group value, respectively.
As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, all 12 fetuses in the two litters that had live fetuses at 1400 mg/kg bw/day had one or more gross, soft tissue and/or skeletal alterations. These alterations included: gross alterations of the tail, limbs and body; soft tissue alteration of the eye, vessels and heart; and skeletal alterations of the skull, vertebral column, limbs, manubrium, sternal centra, clavicle, ribs, and pelvis. The limited number of fetuses and litters with live fetuses compared to the other dosage groups resulted in significantly increased fetal and litter incidences of each of these alterations. At 1400 mg/kg bw/day, the average number of ossification sites per fetus per litter was significantly reduced for caudal vertebrae, sternal centra, xiphoid, metacarpals, phalanges, metatarsals and phalanges. Corresponding to the reduction in fetal weight in the 430 mg/kg bw/day dosage group, ossification site averages for caudal vertebrae, fore- and hindlimb phalanges and metatarsals were also significantly reduced and below the historical control range for this Testing Facility. The number of litters and fetuses with a cervical rib present at the 7th cervical vertebrae was significantly increased at 430 mg/kg bw/day, as compared to the vehicle control group values. No gross lesions related to treatment with Phenethyl Alcohol occurred.
In rats assigned to natural delivery observations, one rat was found dead on DG 13, at 1400 mg/kg bw/day This death was presumed related to treatment with Phenethyl Alcohol because: 1) similar events occurred in the rats assigned to Caesarean-sectioned; and 2) the observation occurred in the high dosage group. All other rats assigned to the natural delivery phase of this study survived to scheduled sacrifice.
Observations consistent with primary irritancy occurred during the gestation period and included: erythema grades 2 and 3, edema grade 2 and flaking grade 3 (1400 mg/kg bw/day); and flaking grades 1 and 2 (≥140 mg/kg bw/day). Each of these skin reactions occurred in an increased or significantly increased number of rats in the 140, 430 and/or 1400 mg/kg bw/day dosage groups, in comparison to the vehicle control group values. The onset and severity of the skin reactions was, in general, dependent on the dosage of Phenethyl Alcohol.
Clinical signs attributed to treatment with Phenethyl Alcohol included: decreased motor activity, vocalization, mild or moderate dehydration (based on skin turgor), ataxia, ptosis, hunched posture, urine-stained abdominal fur, ungroomed coat, red perivaginal substance, grooming/chewing at the back, a red and/or raw appearance to the back: scab forming an open abrasion on the back, an abrasion on the back, lacrimation, scant feces, chromodacryorrhea and soft or liquid feces. The number of affected rats at 1400 mg/kg bw/day during the gestation period was significantly increased, in comparison to the vehicle control group values. In addition, low incidences of impaired righting reflex, chromorhinorrhea, cold to touch, red substance in the cage pan, hyperreactivity to touch, black perinasal substance, mild to moderate dehydration (based on skin turgor) and red substance on the fur occurred at 1400 mg/kg bw/day during the dosage period, and were also attributed to treatment with Phenethyl Alcohol.
Body weight gains were significantly reduced for the entire gestation dosage period and the entire gestation period in the 1400 mg/kg bw/day dosage group, as compared to the vehicle control group values. Body weight gains in the 1400 mg/kg bw/day dosage group were 38% of the vehicle control group value on DGs 7 to 21. The average maternal body weight at 1400 mg/kg bw/day was significantly reduced beginning on DG 8 and continuing until DG 21. At 1400 mg/kg bw/day, the average maternal body weight was 82% of the vehicle control group value on DG 21. In addition, body weight gains were significantly reduced in the 430 and 1400 mg/kg bw/day dosage groups on Days 1 to 4 of lactation. Thereafter, body weight gains were comparable among the dosage groups.
Absolute and relative feed consumption values were reduced or significantly reduced at 1400 mg/kg bw/day on DGs 7 to 21, in comparison to the vehicle control group values. Absolute and relative feed consumption values in the 1400 mg/kg bw/day dosage group were 82% and 94% of the vehicle control group value, respectively, for the entire dosage period. Corresponding to reductions in body weight gain, absolute and relative feed consumption values were also reduced at 1400 mg/kg bw/day dosage group on DLs 1 to 4 and overall for DLs 1 to 14, in comparison to the vehicle control group values.
Pregnancy occurred in 16 to 20 female rats in the four dosage groups. All pregnant dams at 140 and 430 mg/kg bw/day delivered litters, 8 of the 16 pregnant rats delivered a litter at 1400 mg/kg bw/day. Of these 8 dams, one had a litter with no liveborn pups and four had all pups die before day 4 postpartum. At 1400 mg/kg bw/day, the duration of gestation was significantly increased (24.0 days vs. 23.0 days in vehicle controls). In addition, the gestation index at 1400 mg/kg bw/day was significantly reduced, as compared to the vehicle control group value (40% vs. 100% in vehicle controls).
The averages for the total number of pups delivered and the number of liveborn pups were reduced or significantly reduced in this same dosage group, in comparison to the vehicle control group values. The percentage of pups that died or were presumed cannibalized on day 1 and days 2 to 4 postpartum was significantly increased at 1400 mg/kg bw/day, as compared to the vehicle control group values. Reflecting this increase in pup mortality, the viability index at 1400 mg/kg bw/day was significantly reduced relative to the vehicle control group value (63.2% vs. 99.0% in vehicle controls). The average number of surviving pups per litter was significantly reduced at 1400 mg/kg bw/day on days 4, 7, 14 and 21 postpartum, in comparison to the vehicle control group values. In addition, the average pup weight per litter was significantly lower at 1400 mg/kg bw/day on day 1 postpartum relative to the vehicle control group value. On postpartum days 4, 7 and 21, pup body weights at 1400 mg/kg bw/day were 9%, 10% and 7% lower than the corresponding vehicle control group values. At 1400 mg/kg bw/day, a significant number of litters had pups with mild dehydration (based on skin turgor), a thread-like tail and were not nursing.
As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, several of the 24 live pups in the two litters that had live pups at 1400 mg/kg bw/day on postpartum day 21 had a skeletal alteration. The small number of pups and litters resulted in many of these findings occurring at a significantly increased incidence, as compared to the vehicle control group values. These alterations included: short, small or incompletely ossified hyoid ala; a 7th cervical rib; bifid lumbar centrum; misaligned, fused, small or incompletely ossified caudal vertebrae; broad, proximate or short ribs; irregularly shaped manubrium; large sternal centra; irregularly shaped xiphoid or fused metacarpals. The average number of ossification sites per pup per litter was significantly reduced at 1400 mg/kg bw/day for carpals, in comparison to the vehicle control group value. All apparent delays in ossification that were observed in the Caesarean-delivered foetuses at 1400 mg/kg bw/day were resolved by DL21.
The maternal NOAEL was 430 mg/kg/day. The 1400 mg/kg/day dosage caused mortality and reductions in body weight and feed consumption. The developmental NOAEL for pups Caesarean-delivered on day 21 of gestation is 140 mg/kg bw/day. Maternal dosages of 430 and 1400 mg/kg bw/day caused reductions in fetal weight with corresponding delays in fetal skeletal ossification and an increase in the incidence of cervical ribs. All 12 fetuses in the two litters that had live fetuses in the 1400 mg/kg bw/day dosage group had one or more gross, soft tissue and/or skeletal alterations. The 1400 mg/kg bw/day dosage also produced embryo/fetal lethality.
The NOAEL for viability and growth in the offspring naturally delivered and euthanized on day 21 of lactation is 430 mg/kg bw/day. The maternal dosage of 1400 mg/kg bw/day increased the incidence of perinatal mortality. As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, several of the 24 live pups in the two litters that had live pups in the 1400 mg/kg bw/day dosage group on postpartum day 21 had a skeletal alteration. However, all apparent delays in ossification and increased incidences of a cervical rib at the 7th cervical vertebrae that were observed in the Caesarean delivered fetuses in the 430 mg/kg bw/day dosage group were resolved by DL 21.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 July 2008 to 5 February 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Remarks:
- Read-across
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- See overview below
- Principles of method if other than guideline:
- The purpose of this study was to determine the reversibility of skeletal alterations (e.g., rudimentary cervical ribs and vertebral irregularities) and delays in skeletal ossification following treatment of Crl:CD(SD) female rats with Phenethyl Alcohol (PEA) on days 7 through 20 of presumed gestation. One hundred sixty female rats were assigned to four dosage groups, forty rats per sex per group. Each female rat was percutaneously administered the test article, Phenethyl Alcohol (PEA), or the vehicle [reverse osmosis membrane processed deionized water (R.O. water)] once daily beginning on day 7 of presumed gestation and continuing through day 20 of presumed gestation. Due to excessive toxicity, dosage administration for rats in the 1400 mg/kg bw/day dosage group ceased on DGs 15 through 19. Dosages were 0 (Vehicle Control), 140, 430 and 1400 mg/kg bw/day given at a dosage volume of 1.40, 0.14, 0.43 and 1.40 mL/kg, respectively. Twenty female rats per dosage group were Caesarean-sectioned on day 21 of presumed gestation. Twenty female rats per dosage group were allowed to deliver their litters and were sacrificed on day 21 postpartum. The following parameters were evaluated: viability, skin reactions, clinical observations, body weights and body weight gains, feed consumption, Caesarean-sectioning observations, natural delivery observations, necropsy observations, fetal gross external, soft tissue and skeletal alterations, including ossification site averages and pup skeletal alterations, including ossification site averages.
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Millennium Specialty Chemicals, Jacksonville, FL; 8EFN05
- Expiration date of the lot/batch: 25 April 2010
- Purity test date: 99.42%; 01-08-2008
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in a fire proof, tightly closed container at room temperature
- Stability under test conditions: Stability data for the test article formulations used in this study were provided to the Testing Facility by the Sponsor and were not determined during the conduct of this study. - Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 65 days at arrival
- Weight at study initiation: 215 – 250g
- Housing: Stainless steel wire-bottomed cages; bed-o’cobs bedding (The Andersons Industrial Products Group, Maumee, OH) was used as the nesting material.
- Diet: Rats were given ad libitum access to Certified Rodent Diet_ #5002 (PMI_ Nutrition International, St. Louis, MO) in individual feeders.
- Water: Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering access system and/or individual water bottles attached to the cages. Chlorine was added to the processed water as a bacteriostat.
- Acclimation period: After arrival at the Testing Facility, the rats were acclimated to the Elizabethan collars and wrapping (foil application). The collars and wrapping were placed on the rats for an initial acclimation session of one hour. An overnight rest period was provided before the rats were given another acclimation session. Successive acclimation sessions were three hours, six hours and 24-hours in duration. A total of four acclimation sessions were scheduled within the acclimation period.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26°C
- Humidity (%): 30% to 70% (mean 53.6±4.9%)
- Air changes (per hr): Ten changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters.
- Photoperiod (hrs dark / hrs light): An automatically controlled 12-hours light:12-hours dark fluorescent light cycle was maintained.
- Route of administration:
- dermal
- Vehicle:
- water
- Remarks:
- reverse osmosis membrane processed deionized water (R.O. deionized water)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: The test article was considered 100% active/pure for the purpose of dosage calculations. A dosage volume of 0.14, 0.43 and 1.4 mL/kg of a 1000 mg/mL solution was used for a dosage of 140, 430 and 1400 mg/kg bw/day.
VEHICLE
R.O. deionized water is available from a continuous source at the Testing Facility and is maintained at room temperature. - Analytical verification of doses or concentrations:
- no
- Details on analytical verification of doses or concentrations:
- Concentration, homogeneity and stability data for the test article formulations used in this study were provided to the Testing Facility by the Sponsor and were not determined
during the conduct of this study. - Details on mating procedure:
- After acclimation, 194 virgin female rats were placed into cohabitation with breeder male rats, one male rat per female rat. The cohabitation period consisted of a maximum offive days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be DG 0 and assigned to individual housing.
- Duration of treatment / exposure:
- DG 7-20
Due to excessive toxicity, dosage administration for rats in the 1400 mg/kg/day dosage group ceased on DGs 15 through 19 - Frequency of treatment:
- Daily
- Duration of test:
- Caesarean section group: 21 days
Natural Delivery: 42 days (21 days postpartum) - Dose / conc.:
- 140 mg/kg bw/day (nominal)
- Dose / conc.:
- 430 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 400 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Twenty female rats per dosage group were Caesarean-sectioned on day 21 of presumed gestation.
Twenty female rats per dosage group were allowed to deliver their litters and were sacrificed on day 21 postpartum - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
The developmental toxicity of PEA was evaluated in time-mated Crl:COBSCD(SD) BR female rats, 25 per group. Daily doses of 0.14, 0.43 and 1.40 mL/kg [0 (1.40 mL/kg water), 140, 430 and 1400 mg/kg, respectively] of neat PEA were applied to the shaved backs of the rats on DGs 6 through 15 (sperm = DG 0). The 430 mg/kg dose was approximately equivalent to the oral high dose in a previous study (Mankes et al., 1983). To prevent possible oral ingestion of the control (water) or PEA, the dermal application sites were occluded by covering with a fresh piece of aluminum foil that was held in place by a porous adhesive bandage. Observations were made for signs of toxicity, mortality, body weight and feed consumption (DGs 1, 3, 6 and alternate days until necropsy on DG 20). Liver and kidney weights and hematology and serum chemistry parameters were also determined in 15 control and 13 high dose rats on DG 20. In utero development of the conceptuses was assessed by determination of litter values and examination of the fetuses for gross external (all live fetuses), soft tissue (1/2 of the fetuses per litter, using Wilson’s free-hand dissection method) and skeletal changes (1/2 of the fetuses per litter, following alizarin red S-staining).
Maternal toxicity was observed as local irritation at the application site, especially in the high-dose group, but this was considered to be associated with the bandage. Irritability, hunched posture, walking on toes, piloerection, periorbital staining and mortality (1 moribund euthanasia on DG 11 and 2 deaths on DG 13) were observed almost entirely at the 1400 mg/kg dose. These signs started around DG 12, became progressively worse during the remainder of the treatment period, and were essentially absent by termination on DG 20. Mean feed consumption and body weight gains were markedly reduced at the 1400 mg/kg dose. Liver and kidney weights, pregnancy incidences and necropsy observations were unaffected. Developmental toxicity included statistically significant increases in early resorptions, a related reduction in mean litter sizes and a marked reduction in mean fetal weight at the 1400 mg/kg dose. At the 140 mg/kg dose (0.14 mL/kg), no maternal toxicity was observed, and litter parameters were comparable to controls, with the exception of a marginally higher than control incidence of skeletal changes (rudimentary cervical ribs and thoracic vertebral irregularities), observations that showed dose-dependent increases at 430 mg/kg (0.43 mL/kg) and 1400 mg/kg (1.40 mL/kg) doses. Additional findings at 430 mg/kg consisted of occasional fetuses with soft tissue changes similar to those observed at 1400 mg/kg, as well as moderate degrees of incomplete ossification. At 1400 mg/kg, maternal toxicity, significantly increased embryo-fetal death (i.e., resorption), with associated reduced litter size, and significantly reduced fetal body weight were noted. Many morphological changes occurred in 160/161 fetuses at this dose, although more than 40% of the fetuses and 70% of the litters had anophthalmia or microphthalmia, ventricular septal defects, defects or irregularities affecting the thoracic, lumbar and sacrocaudal vertebrae, which were associated with short or kinky tail and defects of the thoracic ribs (Politano et al., 2013).
Based on the results of the study, the maternal NOAEL was 430 mg/kg (0.43 mL/kg) and the developmental NOAEL was approximately 140 mg/kg (0.14 mL/kg). - Maternal examinations:
- F0 Generation Rats
Rats were observed for viability at least twice each day of the study and for clinical observations and general appearance weekly during the acclimation period and on DG 0. The rats were examined for clinical observations, abortions, premature deliveries and deaths before and at approximately hourly intervals for the first four hours after dosage administration and at the end of the normal working day, beginning with the first day of administration (DG 7) and continuing through 13 August 2008 (DGs 5 to 9). Effective 14 August 2008 (DGs 6 to 10), the rats were examined for clinical observations, abortions, premature deliveries and deaths before and one to two hours after dosage administration. During the postdosage period, the rats were examined once daily.
Before the first daily application, and at approximately 24-hour intervals each day thereafter (after rinsing was complete), each skin site was observed for signs of skin irritation and graded using the endpoints described by Draize and the National Research Council. Body weights were recorded weekly during the acclimation period, on DG 0, and daily during the dosage and postdosage periods including prior to sacrifice. Feed consumption values were recorded on DG 0 and were measured and topped-off daily during the dosage period, on DGs 21 and 25 (for dams that did not deliver a litter) and DLs 1, 4, 7, 10 and 14. Because pups begin to consume maternal feed, feed consumption values were not tabulated after DL 14.
Rats assigned to natural delivery were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter sizes (all pups delivered) and pup viability at birth. Maternal behavior was evaluated on DLs 1, 4, 7, 14 and 21. Variations from expected maternal behavior were recorded, if and when present, on all other days of the postpartum period.
Gross lesions and administration sites were retained in neutral buffered 10% formalin for possible future evaluation. Unless specifically cited below, all other tissues were discarded. Representative photographs of maternal gross lesions and fetal gross, soft tissue and skeletal alterations are available in the raw data. - Ovaries and uterine content:
- Rats Assigned to Caesarean-Sectioning
Surviving female rats were euthanized by carbon dioxide asphyxiation on DG 21, Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Uteri of apparently nonpregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites. Uteri and ovaries of apparently nonpregnant rats were retained in neutral buffered 10% formalin and were discarded at the end of the study when authorized by the Study Director. The number and distribution of corpora lutea were recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantation sites, live and dead fetuses and early and late resorptions. An early resorption was defined as one in which organogenesis was not grossly evident. A late resorption was defined as one in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Nonresponding term fetuses were considered to be dead. Dead fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetus was a late resorption. Placentae were examined for size, color and shape.
Rats that died prior to scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. The rats were examined for gross lesions. The lungs, trachea and esophagus were perfused and saved in neutral buffered 10% formalin for possible future evaluation. The administration site, heart, liver, kidneys, stomach and spleen were retained in neutral buffered 10% formalin for possible histological evaluation. Pregnancy status and uterine contents of female rats was recorded. Conceptuses in utero were examined to the extent possible, using the same methods described for term fetuses. The uteri of the nonpregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites. The uteri and ovaries of the nonpregnant rats were retained in neutral buffered 10% formalin and were discarded at the end of the study when authorized by the Study Director.
Rats Assigned to Natural Delivery
After completion of the 21-day postpartum period, female rats were sacrificed by carbon dioxide asphyxiation and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The number and distribution of implantation sites was recorded. Rats that did not deliver a litter were sacrificed on DG 25 and examined for gross lesions. Uteri were examined while being pressed between glass plates, to confirm the absence of implantation sites. Uteri and ovaries of nonpregnant rats were retained in neutral buffered 10% formalin and were discarded when authorized by the Study Director. Dams with no surviving pups were sacrificed after the last pup was found dead or missing, presumed cannibalized. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The rat that died before scheduled termination was examined for the cause of death after the observation was made. The rat was examined for gross lesions. The lungs, trachea and esophagus were perfused and saved in neutral buffered 10% formalin for possible future evaluation. The administration site, heart, liver, kidneys, stomach and spleen were retained in neutral buffered 10% formalin for possible histological evaluation. Pregnancy status and uterine contents of rat was recorded. Conceptuses in utero were examined to the extent possible, using the same methods described for term fetuses. - Fetal examinations:
- F1 Generation
PPD 1 was defined as the day of birth and was also the first day on which all pups in a litter were individually weighed (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam). Litters were not culled during the lactation period because random selection of pups for culling could result in potential biases in pup viabilities and body weight gained during this period. Each litter was evaluated for viability at least twice daily. The pups in each litter were counted once daily. Clinical observations were recorded once daily. Pup body weights were recorded on DLs 1, 4, 7, 14 and 21 (prior to sacrifice).
Rats Assigned to Caesarean-Sectioning
Each fetus was removed from the uterus, placed in an individual container and individually identified with a tag noting the study number, litter number, uterine distribution and fixative. Each fetus was subsequently weighed and examined for sex and gross external alterations. Live fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbital. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations, using a variation of the microdissection technique of Staples10,11. These fetuses were then fixed in Bouin's solution and the heads were subsequently examined by free-hand sectioning; head sections were stored in alcohol. The decapitated carcasses were discarded. The remaining fetuses (approximately one-half of the fetuses in each litter) were eviscerated, cleared, stained with alizarin red S12 and examined for skeletal alterations. The fetuses were initially fixed in alcohol. Skeletal preparations were retained in glycerin with thymol added as a preservative.
F1 Generation Pups
Pups that died before initial examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered liveborn and to have died shortly after birth. Pups were preserved in alcohol for possible future evaluation. Pups found dead on days 2 to 21 postpartum were examined for gross lesions and for the cause of death. Pups were eviscerated, skinned and preserved in alcohol for possible future evaluation. After completion of the 21-day postpartum period, surviving F1 Generation pups were sacrificed by carbon dioxide asphyxiation. Pups were tagged with identification noting the study number, litter number, and fixative. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Necropsy of the pups included a single cut at the suture of the frontal and parietal bones of the skull, and the cross-sectioned brain was examined for hydrocephaly. Following gross necropsy evaluations, all pups were eviscerated and skinned. The pups were examined for skeletal alterations after staining with alizarin red S12. The pups were initially fixed in alcohol; skeletal preparations were retained in glycerin with thymol added as a preservative. The gross lesion observed was preserved in neutral buffered 10% formalin. Representative photographs of skeletal alterations are available in the raw data. - Statistics:
- Averages and percentages were calculated. Clinical observations and other proportional data were analyzed, using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g., maternal body weights, body weight changes, feed consumption values, pup body weights and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and fetal anomaly data) were analyzed, using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test was not significant (p >0.001)]. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test1was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s
Test was significant (p≤0.001)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p≤ 0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there weregreater than 75% ties, Fisher’s Exact Test was used to analyze the data. Count data were evaluated, using the procedures described above for the Kruskal-Wallis Test. - Historical control data:
- Provided for period January 2006 - 2008 (Appendix 5)
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Rats Assigned To Caesarean-Sectioning
Clinical signs attributed to treatment with Phenethyl Alcohol that achieved statistical significance are summarized in Text Table 2. The number of affected rats in the 1400 mg/kg/day dosage group was significantly increased (p_ 0.01) in comparison to the vehicle control group values. In addition, low incidences of respiratory distress (i.e, labored breathing, bradypnea, dyspnea, tachypnea and gasping), pale extremities, pale appearance, cold to touch, soft or liquid feces, limited use of both hindlimbs, circling to the right, prostration, red substance in the cage pan, lacrimation, irritation on the back (i.e., red discoloration, irritated and/or raw), grooming or chewing at the back, thrashing in the nesting box, rigid posture, irregular breathing, mild to moderate dehydration (based on skin turgor), dehydration without a severity and mucoid feces occurred in the 1400 mg/kg/day dosage group during the dosage period. The occurrence of these clinical signs did not differ significantly from the vehicle control group value (p> 0.05). However, these observations were also attributed to treatment with Phenethyl Alcohol because: 1) the clinical signs occurred in rats that had concomitant signs of clinical toxicity; and/or 2) the observations occurred in rats that died or were humanely euthanized prior to scheduled sacrifice. All other clinical observations were considered unrelated to percutaneous administration of Phenethyl Alcohol because: 1) the incidences were not dosage-dependent; and 2) the number of affected rats in a group was not considerably greater than the number affected in the vehicle control group. These clinical observations included slight excess salivation, sparse hair coat (limbs and/or underside), portion of the tail missing and missing/broken incisors. Fewer rats (significant at p_ 0.01) in the 140, 430 and 1400 mg/kg/day dosage group had a swollen neck or head, in comparison to the vehicle control group incidence. This finding was not attributed to percutaneous administration of Phenethyl Alcohol because the expected toxicological effect would be an increase in the observation rather than a decrease (Summaries - Tables A1 and A13) - Dermal irritation (if dermal study):
- effects observed, treatment-related
- Description (incidence and severity):
- Rats Assigned To Caesarean-Sectioning
Observations consistent with primary irritancy occurred in female rats percutaneously
administered Phenethyl Alcohol during the gestation period (DGs 7 through 21). The
onset and severity of the skin reactions was, in general, dependent on the dosage of
Phenethyl Alcohol.
Erythema grades 1 (barely perceptible redness) and 2 (distinct redness) occurred in more
rats (significant at p≤ 0.01) in the 1400 mg/kg bw/day dosage group, in comparison to the
vehicle control group values. Two rats in the 1400 mg/kg bw/day dosage group also had an
observation of erythema grade 3 (“beet red” color) on one or three occasions during the
dosage period; no other rat had this skin reaction. Significantly more (p≤0.01) rats in the
1400 mg/kg bw/day dosage group also had flaking grades 2 (distinct scales) and
3 (pronounced flaking with denuded sites) than rats in the corresponding vehicle control
group. In addition, flaking grade 2 was observed in a significant number of rats (p≤ 0.01)
in the 430 mg/kg bw/day dosage group, in comparison to the vehicle control group values.
All other skin reactions occurred at a comparable incidence relative to the vehicle control
group value and did not differ significantly. Flaking grade 1 (barely perceptible scales)
and scabbing was observed in each dosage group, including the vehicle control group.
One rat in each of the vehicle control and 1400 mg/kg bw/day dosage group had edema
grade 1 (mild, raised <1 mm) on one and three occasions, respectively. (Summaries - Tables A1 and A13) - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Rats Assigned To Caesarean-Sectioning
Mortality was significantly increased (p≤0.01) in the 1400 mg/kg/day dosage group, in comparison to the vehicle control group value. Five rats (significant at p≤0.01) were found dead in the 1400 mg/kg/day dosage group, and three rats (significant at p≤0.01) in this same dosage group were humanely euthanized. These deaths occurred after 3 to 11 dosages of the test article had been administered percutaneously. As shown in Text Table 1, most of these rats had decreased motor activity, impaired or lost righting reflex, ataxia, hunched posture, mild or moderate dehydration (based on skin turgor), red perivaginal substance and vocalization before death. In addition, observations consistent with primary irritancy (erythema and flaking) occurred in most of the female rats that died or were euthanized. These skin reactions and clinical signs are consistent with observations noted in rats that survived to scheduled sacrifice; therefore, the deaths were attributed to treatment with Phenethyl Alcohol. There were no common gross lesions observed in these rats. Skin reactions, clinical and necropsy observations for these rats are provided in Text Table 1. All other rats assigned to the Caesarean-sectioning phase of this study survived to scheduled sacrifice on DG 21 9 (Summaries - Tables A1 and A13). - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Rats Assigned To Caesarean-Sectioning
Body weight losses occurred in each dosage group, including the vehicle control group,
during the first three days of the dosage period (DGs 7 to 10). At 1400 mg/kg bw/day, the
body weight losses reached statistical significance relative to the vehicle control group
value (p≤0.01), and continued to persist on DGs 10 to 14 and 14 to 17. Thereafter, body
weight gains were significantly reduced (p≤0.01) in the 1400 mg/kg bw/day dosage group,
in comparison to the vehicle control group value (44% lower than vehicle control value
on DGs 17 to 21). Overall, a net loss in body weight (significant at p≤0.01) occurred in
the 1400 mg/kg bw/day dosage group for the entire dosage period (calculated as DGs 7 to
21), while body weight gains were significantly reduced (p≤0.01) for the entire gestation
period (DGs 0 to 21), as compared to the vehicle control group values.
Reflecting the effects of Phenethyl Alcohol on body weight gains, the average maternal
body weight in the 1400 mg/kg bw/day dosage group was significantly reduced (p≤0.01)
beginning on DG 8 and continuing until DG 21. The average maternal body weight on
DG 21 was 100%, 100% and 71% of the vehicle control group value in the 140, 430 and
1400 mg/kg bw/day dosage groups, respectively.
Body weights and body weight gains were unaffected by dosages of Phenethyl Alcohol
as high as 430 mg/kg bw/day. (Figure 1; Summaries - Tables A2 and A3) - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Rats Assigned To Caesarean-Sectioning
Absolute feed consumption values were significantly reduced (p≤0.01) in the
1400 mg/kg bw/day dosage group for the entire dosage period (calculated as DGs 7 to 21)
and at all tabulated intervals within the dosage period, in comparison to the vehicle
control group values. In addition, absolute feed consumption values were significantly
reduced (p≤0.01) at 1400 mg/kg bw/day for the entire gestation period (DGs 0 to 21), as
compared to the vehicle control group value. Absolute feed consumption values in the
140, 430 and 1400 mg/kg bw/day dosage groups were 102%, 101% and 78% of the vehicle
control group value, respectively, for the entire dosage period (calculated as DGs 7 to
21).
Relative to body weight, gestating rats in the 1400 mg/kg bw/day dosage group consumed
less or significantly less (p≤0.01) feed on DGs 7 to 10, DGs 10 to 14 and DGs 14 to 17,
in comparison to the vehicle control group values. Overall, relative feed consumption in
the 1400 mg/kg bw/day was reduced by 6% for the entire dosage period (calculated as DGs 7
to 21), as compared to the vehicle control group value. Absolute and relative feed consumption values were unaffected by dosages of Phenethyl Alcohol as high as 430 mg/kg bw/day. (Summaries - Tables A4 and A5) - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- 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:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Rats Assigned To Caesarean-Sectioning
No gross lesions related to treatment with Phenethyl Alcohol occurred. All necropsy
observations were considered unrelated to Phenethyl Alcohol because: 1) the
observations were not dosage-dependent; 2) the observations occurred in only one rat in
any dosage group; and/or 3) the observation occurred in a rat that died prior to scheduled
sacrifice. These gross lesions included a mottled appearance (red and dark red) to all
lobes of the lungs and the presence of two fetuses at one implantation site. No other
gross lesions occurred. (Summary - Table A13) - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Total litter losses by resorption:
- effects observed, treatment-related
- Description (incidence and severity):
- Rats Assigned To Caesarean-Sectioning
Pregnancy occurred in 16 to 20 rats in each dosage group. As a result of the mortality (i.e., found dead and unscheduled sacrifice) that occurred in the 1400 mg/kg bw/day dosage group, Caesarean-sectioning observations on DG 21 were based on 20, 18, 20 and 9 pregnant rats in the 0 (Vehicle Control), 140, 430 and 1400 mg/kg bw/day dosage groups, respectively. Postimplantation loss (i.e., early and late resorptions and the percentage of resorbed conceptuses per litter) was increased or significantly increased (p≤0.01) in the 1400 mg/kg bw/day dosage group, in comparison to the vehicle control group values. The average for total resorptions in the 1400 mg/kg bw/day dosage group was 12.6 per litter relative to 1.0 per litter in the vehicle control group. Seven of the nine rats in the 1400 mg/kg bw/day dosage group that were Caesarean-sectioned on DG 21 had total litter loss (i.e., 100% resorbed conceptuses; significant at p≤0.01). As a result, fewer dams (significant at p≤0.01) had litters with viable fetuses, in comparison to the vehicle control group value. These increases in postimplantation loss resulted in an overall reduction (significant at p≤0.01) in the averages for litter size and live fetuses in the 1400 mg/kg bw/day dosage group, in comparison to the vehicle control group values (1.3 per litter vs. 14.6 per litter in vehicle controls).
The average fetal body weight (total, male and female) in the 430 and 1400 mg/kg bw/day dosage groups was significantly lower (p≤0.01) than the concurrent vehicle control group values. These values were also below the range observed historically at the Testing Facility. The combined fetal weight in the 430 and 1400 mg/kg bw/day dosage groups was 91% and 48% of the vehicle control group value, respectively.
No other Caesarean-sectioning or litter parameters were affected by dosages of Phenethyl Alcohol as high as 1400 mg/kg bw/day. The litter averages for corpora lutea, implantations, and the percentage of live male fetuses were comparable among the four dosage groups and did not significantly differ. There were no dead fetuses. All placentae appeared normal. (Summaries - Tables A6 and A7) - Dose descriptor:
- NOAEL
- Effect level:
- 430 mg/kg bw/day (nominal)
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- mortality
- Abnormalities:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- Rats Assigned To Caesarean-Sectioning
Fetal Alterations
Fetal alterations were defined as: 1) malformations (irreversible changes that occur at low incidences in this species and strain); or 2) variations (common findings in this species and strain and reversible delays or accelerations in development). Litter averages were calculated for specific fetal ossification sites as part of the evaluation of the degree of fetal ossification. Fetal evaluations were based on 292, 266, 296 and 12 live DG 21 Caesarean-delivered fetuses in 20, 18, 20 and 2 litters in the 0 (Vehicle Control), 140, 430 and 1400 mg/kg bw/day dosage groups, respectively. Each of these fetuses was examined for gross external alterations. Of these respective fetuses, 142, 130, 143 and 6 fetuses were examined for soft tissue alterations, and 150, 136, 153 and 6 fetuses were examined for skeletal alterations and fetal ossification site averages. Dams in the 1400 mg/kg bw/day dosage group exhibited extensive maternal and embryo/fetal toxicity. All 12 fetuses in the two litters that had live fetuses in this dosage group had one or more gross, soft tissue and/or skeletal alterations. The average number of ossification sites per fetus per litter was significantly reduced (p≤0.01) in the 1400 mg/kg bw/day dosage group for caudal vertebrae, sternal centra, ossified xiphoid, metacarpals, forelimb phalanges, metatarsals and hindlimb phalanges. The number of litters and fetuses with a cervical rib present at the 7th cervical vertebrae was significantly increased (p≤0.01) in this dosage group. Corresponding to the reduction in fetal weight in the 430 mg/kg bw/day dosage group, ossification site averages for caudal vertebrae, fore- and hindlimb phalanges and metatarsals were significantly reduced (p≤0.01) and below the historical control range for this Testing Facility. The number of litters and fetuses with a cervical rib present at the 7th cervical vertebrae was significantly increased (p≤0.01 and p≤0.05, respectively) in this dosage group. No gross external, soft tissue or skeletal fetal alterations (malformations or variations) were caused by dosages of the test article as high as 140 mg/kg bw/day. Fetal ossification site averages in the 140 mg/kg bw/day dosage group were comparable to the vehicle control group values. (Summaries - Tables A8 through A12)
Summary of Fetal Alterations
In the 0 (Vehicle Control), 140, 430 and 1400 mg/kg bw/day dosage groups, litters with fetuses with alterations numbered 6 (30.0%), 6 (33.3%), 12 (60.0%) and 2 (100.0%), respectively. The numbers of fetuses with any alteration observed were 6 (2.0%), 6 (2.2%), 23 (7.8%)* and 12 (100.0%)**, and the percentages of fetuses with any alteration per litter were 2.0%, 2.1%, 7.8%* and 100.0%** in these same respective dosage groups. As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, all 12 fetuses in the two litters that had live fetuses in the 1400 mg/kg bw/day dosage group on DG 21 had one or more gross, soft tissue and/or skeletal alterations. These alterations included gross alterations of the tail (absent, short, thread-like and bent), limbs (absent digits and flexed forepaws and rotated hindlimbs) and body (whole edema); soft tissue alteration of the eye (microphthalmia), vessels (constricted pulmonary artery and constricted aorta) and heart (interventricular septal defect); and skeletal alterations of the skull (incompletely ossified tympanic rings and eye socket small), vertebral column (7th cervical rib, bifid centrum in one or more thoracic and/or lumbar vertebrae, incompletely ossified arches, fused arches, not ossified arches, close-set arches, fused centra, unilaterally ossified centra in one or more cervical, thoracic, lumbar, sacral and/or caudal vertebrae), limbs (irregularly shaped metatarsals or humerus, non-ossified metatarsals, absent digits, absent or fused phalanges, bent radius, bent ulna, bent humerus, bent femur, short femur, irregular number of metacarpals or metatarsals present), manubrium (duplicated or small), sternal centra (incompletely ossified or duplicated), clavicle (bent), and ribs (wavy, short, bowed or fused), pelvis (close-set ilium, small ischium, small or not ossified pubes). The limited number of fetuses and litters with live fetuses compared to the other dosage groups resulted in significantly increased (p≤0.01) fetal and litter incidences of each of these alterations. The average number of ossification sites per fetus per litter was significantly reduced (p_ 0.01) in the 1400 mg/kg bw/day dosage group for caudal vertebrae, sternal centra, ossified xiphoid, metacarpals, phalanges, metatarsals and phalanges. None of these fetuses will be discussed further. Individual observations for these fetuses are available in Table A20. All other fetal alterations (malformations and variations) that occurred in the 0 (Vehicle Control), 140 and 430 mg/kg bw/day dosage groups are summarized in the following text. (Summary - Table A8)
Fetal Gross External Alterations Fetus 7119-16 in the 0 (Vehicle Control) group had a cleft palate and a short digit (1st) on the right hindpaw. Skeletal evaluation confirmed the cleft palate and revealed anomalies of the thoracic vertebrae (fused arches, a small arch and not ossified and unilaterally ossified centra), ribs (fused and not ossified) and hindlimbs (small phalanx). Fetus 7135-12 in the 140 mg/kg bw/day dosage group had whole body edema. No other alterations occurred in this fetus. Fetus 7150-2 in the 430 mg/kg bw/day dosage group had an absent tail. This fetus had no other alterations. No other gross external fetal alterations occurred in the 0 (Vehicle Control), 140 or 430 mg/kg bw/day dosage groups. (Summary - Table A9)
Fetal Soft Tissue Alterations No soft tissue fetal alterations (malformations or variations) occurred in the 0 (Vehicle Control), 140 or 430 mg/kg bw/day dosage groups. (Summary - Table A10)
Fetal Skeletal Alterations
Malformations-Skull Fetus 7119-16 in the 0 (Vehicle Control) had an incompletely ossified palate and other alterations, as previously described.
Malformations-Vertebrae/Ribs Fetus 7119-16 in the 0 (Vehicle Control) mg/kg bw/day dosage group and fetus 7137-3 in the 140 mg/kg bw/day dosage group had fused arches in one or more thoracic vertebrae. Fetus 7119-16 had other alterations as previously described. Fetus 7137-3 also had an irregularly shaped manubrium, asymmetric and incompletely sternal centra, an irregularly shaped xiphoid, a hemivertebra (arch and centrum with an attached rib) present as a thoracic vertebra, a small arch in one or more thoracic vertebrae, bifid centra in one or more thoracic vertebrae, fused centra in one or more thoracic vertebrae and fused ribs. Fetus 7119-16 in the 0 (Vehicle Control) mg/kg bw/day dosage group had a small arch and a not ossified centrum in a thoracic vertebra. This fetus had other alterations as previously described. One other fetus in the 140 mg/kg bw/day dosage group (fetus 7123-6) had a hemivertebra (arch and centrum with an attached rib) present as a thoracic vertebra. This fetus also had a small arch in a thoracic vertebra, a bifid centrum in a thoracic vertebra, a unilaterally ossified centrum in a thoracic vertebra and proximate ribs. Fetus 7119-16 in the vehicle control group had a not ossified rib, as previously described.
Malformations-limbs Fetus 7119-16 in the 0 (Vehicle Control) mg/kg bw/day dosage group had a small phalanx (distal 3rd) on both hindlimbs. This fetus had additional alterations as previously described. No other skeletal malformations occurred.
Variations - Skull Fetus 7143-1 in the 430 mg/kg bw/day dosage group had a large frontal and nasal-frontal suture. This fetus had no additional alterations.
Variations - Vertebrae A total of 2 (7107-1 and 7113-4), 1 (7125-7) and 5 (7142-1, -11; 7146-9; 7147-1, -4) fetuses from 2, 1 and 3 litters in the 0 (Vehicle Control), 140 and 430 mg/kg bw/day dosage groups, respectively, had an arch in the 6th cervical vertebra that had the appearance (size and shape) of the arch in the 7th cervical vertebra. No additional alterations occurred in these fetuses. Fetus 7159-7 had an incompletely ossified arch in a cervical vertebra. This fetus had no additional alterations. A bifid centrum in one or more thoracic vertebrae occurred in 3 (7105-1, 7116-9 and 7117-4), 3 (7123-6, 7133-14 and 7137-3) and 2 (7142-9 and 7158-9) fetuses each from a different litter in the 0 (Vehicle Control), 140 and 430 mg/kg bw/day dosage groups, respectively. Of these fetuses, several had additional alterations. Fetus 7116-9 in the vehicle control group also had wavy ribs and fetus 7117-4 in the vehicle control group also had a bifid centrum in a lumbar vertebra. Fetuses 7123-6 and 7137-3 in the 140 mg/kg bw/day dosage group also had other alterations, as previously described. No additional alterations occurred in these fetuses. Fetus 7119-16 in the 0 (Vehicle Control) mg/kg bw/day dosage group and fetus 7123-6 in the 140 mg/kg bw/day dosage group each had unilateral ossification of the centrum in a thoracic vertebra. These fetuses had other alterations, as previously described.
Variations -Ribs A cervical rib at the 7th cervical vertebra, a common variation in this strain of rat20, was present in 0, 1 and 12* fetuses from 0, 1 and 6** litters in the 0 (Vehicle Control), 140 and 430 mg/kg bw/day dosage groups, respectively. None of these fetuses had any additional alterations.
Variations - Sternum Fetus 7137-3 in the 140 mg/kg bw/day dosage group had an irregularly shaped manubrium and xiphoid, in addition to incompletely ossified and asymmetric sternal centra. This fetus had other alterations as previously described. Fetus 7160-1 in the 430 mg/kg bw/day dosage group had a not ossified 1st sternal centrum. No other alterations occurred in this fetus. No other skeletal fetal alterations occurred in the 0 (Vehicle Control), 140 or 430 mg/kg bw/day dosage groups.
Fetal Ossification Site Averages The average numbers of ossified caudal vertebrae, forelimb phalanges and hindlimb metatarsals and phalanges were significantly reduced (p< 0.01) in the 430 and 1400 mg/kg bw/day dosage groups, as compared with the vehicle control group values. In addition, the average number of ossified sternal centers, xiphoids and forelimb metacarpals were significantly reduced (p≤0.01) in the 1400 mg/kg bw/day dosage group, in comparison to the vehicle control group values. Each of the average litter values were below the historical range of the Testing Facility, as shown in the table below. These delays in ossification correspond with the reductions in fetal body weights that occurred in the 430 and 1400 mg/kg bw/day dosage groups, as previously described. There were no other statistically significant or biologically important differences among the four dosage groups in the average numbers of ossification sites per fetus for the hyoid, vertebrae (cervical, thoracic, lumbar and sacral), ribs, sternum (manubrium), forelimbs (carpals) or hindlimbs (tarsals). (Summaries - Tables A11 and A12).
* Significantly different from the vehicle control group value (p≤0.05).
** Significantly different from the vehicle control group value (p≤ 0.01). - Dose descriptor:
- NOAEL
- Remarks:
- Developmental (DG 21)
- Effect level:
- 140 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- fetal/pup body weight changes
- skeletal malformations
- Dose descriptor:
- NOAEL
- Remarks:
- viability and growth on day 21 of lactation
- Effect level:
- 430 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- reduction in number of live offspring
- skeletal malformations
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- skeletal: rib
- Description (incidence and severity):
- All apparent delays in ossification and increased incidences of a cervical rib at the 7th cervical vertebrae that were observed in the Caesareandelivered fetuses in the 430 mg/kg bw/day dosage group were resolved by DL 21.
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 140 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- no
- Conclusions:
- In a Combined Dermal Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PEA (phenethyl alcohol) in Rats, the maternal NOAEL is 430 mg/kg/day. The 1400 mg/kg/day dosage caused mortality and reductions in body weight and feed consumption. The developmental NOAEL for pups Caesarean-delivered on day 21 of gestation is 140 mg/kg/day. Maternal dosages of 430 and 1400 mg/kg/day caused reductions in fetal weight with corresponding delays in fetal skeletal ossification and an increase in the incidence of cervical ribs. All 12 fetuses in the two litters that had live fetuses in the 1400 mg/kg/day dosage group had one or more gross, soft tissue and/or skeletal alterations. The 1400 mg/kg/day dosage also produced embryo/fetal lethality. The NOAEL for viability and growth in the offspring naturally delivered and euthanized on day 21 of lactation is 430 mg/kg/day. The maternal dosage of 1400 mg/kg/day increased the incidence of perinatal mortality. As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, several of the 24 live pups in the two litters that had live pups in the 1400 mg/kg/day dosage group on postpartum day 21 had a skeletal alteration. However, all apparent delays in ossification and increased incidences of a cervical rib at the 7th cervical vertebrae that were observed in the Caesarean delivered fetuses in the 430 mg/kg/day dosage group were resolved by DL 21.
- Executive summary:
In a combined dermal developmental and perinatal/postnatal reproduction toxicity study (58462) PEA (99.42%) or deionized water was administered dermally (occlusive) to 40 female Crl:CD(SD) rats at dose levels of 0, 140, 430 and 1400 mg/kg bw/day beginning on day 7 of presumed gestation and continuing through day 20 of presumed gestation. Twenty rats per dosage group were Caesarean-sectioned on day 21 of presumed gestation and 20 rats per dosage group were allowed to deliver their litters and were sacrificed on day 21 postpartum. Skin irritation was assessed using the Draize method.
In the rats assigned to caesarean-sectioning, mortality was significantly increased at 1400 mg/kg bw/day, in comparison to the vehicle control group value. Five and three rats in the 1400 mg/kg bw/day dosage group were found dead or humanely euthanized, respectively. Most of these rats had decreased motor activity, impaired or lost righting reflex, ataxia, hunched posture, mild or moderate dehydration (based on skin turgor), red perivaginal substance and vocalization before death. In addition, observations consistent with primary irritancy (erythema and flaking) occurred in most of the female rats that died or were euthanized. These skin reactions and clinical signs are consistent with observations noted in rats that survived to scheduled sacrifice; therefore, the deaths were attributed to treatment with Phenethyl Alcohol.
Observations consistent with primary irritancy occurred in female rats percutaneously administered Phenethyl Alcohol during the gestation period. These skin reactions included: flaking grade 2 (≥430 mg/kg bw/day) and erythema grades 1 through 3 and flaking grade 3 (1400 mg/kg bw/day). Each of these skin reactions, with the exception of erythema grade 3, occurred in significantly more rats in the 430 and/or 1400 mg/kg bw/day dosage groups, in comparison to the vehicle control group values. The onset and severity of the skin reactions was, in general, dependent on the dosage of Phenethyl Alcohol.
Clinical signs attributed to treatment with Phenethyl Alcohol included: decreased motor activity, impaired righting reflex, lost righting reflex, ataxia, hunched posture, ptosis, hyperreactivity to touch and/or in the nesting box, vocalization,mild, moderate or severe dehydration (based on skin turgor), cold extremities, moderate excess salivation, chromorhinorrhea, chromodacryorrhea, urine-stained abdominal fur, ungroomed coat, scab forming an abrasion on the back, clear and/or red perivaginal substance and scant feces. The number of affected rats at 1400 mg/kg bw/day was significantly increased in comparison to the vehicle control group values.
A statistically significant loss in body weight, coupled with reduced or significantly reduced feed consumption values, occurred at 1400 mg/kg bw/day for the entire dosage period, as compared to the vehicle control group values. Absolute and relative feed value, respectively, for DGs 7 to 21. Overall, body weight gains and absolute feed consumption values were significantly reduced for the entire gestation period, as compared to the vehicle control group values. At 1400 mg/kg bw/day, the average maternal body weight was significantly reduced beginning on DG 8 and continuing until DG 21.
Pregnancy occurred in 16 to 20 rats in each dosage group. As a result of the increased mortality that occurred in the 1400 mg/kg bw/day dosage group, Caesarean-sectioning observations on DG 21 were based on 20, 18, 20 and 9 pregnant rats in the 0 (Vehicle Control), 140, 430 and 1400 mg/kg bw/day dosage groups, respectively. At 1400 mg/kg bw/day, postimplantation loss (i.e., early and late resorptions and the percentage of resorbed conceptuses per litter) was increased or significantly increased, in comparison to the vehicle control group values. Seven of the nine rats in this dosage group had total litter loss (i.e., 100% resorbed conceptuses). These increases in postimplantation loss resulted in an overall significant reduction in the averages for litter size and live fetuses at 1400 mg/kg bw/day, in comparison to the vehicle control group values (1.3 per litter vs. 14.6 per litter in vehicle controls). At ≥430 mg/kg bw/day, the average fetal body weight (total, male and female) was significantly lower than the concurrent vehicle control group values. These values were also below the range observed historically at the Testing Facility. The combined fetal weight at 430 and 1400 mg/kg bw/day was 91% and 48% of the vehicle control group value, respectively.
As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, all 12 fetuses in the two litters that had live fetuses at 1400 mg/kg bw/day had one or more gross, soft tissue and/or skeletal alterations. These alterations included: gross alterations of the tail, limbs and body; soft tissue alteration of the eye, vessels and heart; and skeletal alterations of the skull, vertebral column, limbs, manubrium, sternal centra, clavicle, ribs, and pelvis. The limited number of fetuses and litters with live fetuses compared to the other dosage groups resulted in significantly increased fetal and litter incidences of each of these alterations. At 1400 mg/kg bw/day, the average number of ossification sites per fetus per litter was significantly reduced for caudal vertebrae, sternal centra, xiphoid, metacarpals, phalanges, metatarsals and phalanges. Corresponding to the reduction in fetal weight in the 430 mg/kg bw/day dosage group, ossification site averages for caudal vertebrae, fore- and hindlimb phalanges and metatarsals were also significantly reduced and below the historical control range for this Testing Facility. The number of litters and fetuses with a cervical rib present at the 7th cervical vertebrae was significantly increased at 430 mg/kg bw/day, as compared to the vehicle control group values. No gross lesions related to treatment with Phenethyl Alcohol occurred.
In rats assigned to natural delivery observations, one rat was found dead on DG 13, at 1400 mg/kg bw/day This death was presumed related to treatment with Phenethyl Alcohol because: 1) similar events occurred in the rats assigned to Caesarean-sectioned; and 2) the observation occurred in the high dosage group. All other rats assigned to the natural delivery phase of this study survived to scheduled sacrifice.
Observations consistent with primary irritancy occurred during the gestation period and included: erythema grades 2 and 3, edema grade 2 and flaking grade 3 (1400 mg/kg bw/day); and flaking grades 1 and 2 (≥140 mg/kg bw/day). Each of these skin reactions occurred in an increased or significantly increased number of rats in the 140, 430 and/or 1400 mg/kg bw/day dosage groups, in comparison to the vehicle control group values. The onset and severity of the skin reactions was, in general, dependent on the dosage of Phenethyl Alcohol.
Clinical signs attributed to treatment with Phenethyl Alcohol included: decreased motor activity, vocalization, mild or moderate dehydration (based on skin turgor), ataxia, ptosis, hunched posture, urine-stained abdominal fur, ungroomed coat, red perivaginal substance, grooming/chewing at the back, a red and/or raw appearance to the back: scab forming an open abrasion on the back, an abrasion on the back, lacrimation, scant feces, chromodacryorrhea and soft or liquid feces. The number of affected rats at 1400 mg/kg bw/day during the gestation period was significantly increased, in comparison to the vehicle control group values. In addition, low incidences of impaired righting reflex, chromorhinorrhea, cold to touch, red substance in the cage pan, hyperreactivity to touch, black perinasal substance, mild to moderate dehydration (based on skin turgor) and red substance on the fur occurred at 1400 mg/kg bw/day during the dosage period, and were also attributed to treatment with Phenethyl Alcohol.
Body weight gains were significantly reduced for the entire gestation dosage period and the entire gestation period in the 1400 mg/kg bw/day dosage group, as compared to the vehicle control group values. Body weight gains in the 1400 mg/kg bw/day dosage group were 38% of the vehicle control group value on DGs 7 to 21. The average maternal body weight at 1400 mg/kg bw/day was significantly reduced beginning on DG 8 and continuing until DG 21. At 1400 mg/kg bw/day, the average maternal body weight was 82% of the vehicle control group value on DG 21. In addition, body weight gains were significantly reduced in the 430 and 1400 mg/kg bw/day dosage groups on Days 1 to 4 of lactation. Thereafter, body weight gains were comparable among the dosage groups.
Absolute and relative feed consumption values were reduced or significantly reduced at 1400 mg/kg bw/day on DGs 7 to 21, in comparison to the vehicle control group values. Absolute and relative feed consumption values in the 1400 mg/kg bw/day dosage group were 82% and 94% of the vehicle control group value, respectively, for the entire dosage period. Corresponding to reductions in body weight gain, absolute and relative feed consumption values were also reduced at 1400 mg/kg bw/day dosage group on DLs 1 to 4 and overall for DLs 1 to 14, in comparison to the vehicle control group values.
Pregnancy occurred in 16 to 20 female rats in the four dosage groups. All pregnant dams at 140 and 430 mg/kg bw/day delivered litters, 8 of the 16 pregnant rats delivered a litter at 1400 mg/kg bw/day. Of these 8 dams, one had a litter with no liveborn pups and four had all pups die before day 4 postpartum. At 1400 mg/kg bw/day, the duration of gestation was significantly increased (24.0 days vs. 23.0 days in vehicle controls). In addition, the gestation index at 1400 mg/kg bw/day was significantly reduced, as compared to the vehicle control group value (40% vs. 100% in vehicle controls).
The averages for the total number of pups delivered and the number of liveborn pups were reduced or significantly reduced in this same dosage group, in comparison to the vehicle control group values. The percentage of pups that died or were presumed cannibalized on day 1 and days 2 to 4 postpartum was significantly increased at 1400 mg/kg bw/day, as compared to the vehicle control group values. Reflecting this increase in pup mortality, the viability index at 1400 mg/kg bw/day was significantly reduced relative to the vehicle control group value (63.2% vs. 99.0% in vehicle controls). The average number of surviving pups per litter was significantly reduced at 1400 mg/kg bw/day on days 4, 7, 14 and 21 postpartum, in comparison to the vehicle control group values. In addition, the average pup weight per litter was significantly lower at 1400 mg/kg bw/day on day 1 postpartum relative to the vehicle control group value. On postpartum days 4, 7 and 21, pup body weights at 1400 mg/kg bw/day were 9%, 10% and 7% lower than the corresponding vehicle control group values. At 1400 mg/kg bw/day, a significant number of litters had pups with mild dehydration (based on skin turgor), a thread-like tail and were not nursing.
As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, several of the 24 live pups in the two litters that had live pups at 1400 mg/kg bw/day on postpartum day 21 had a skeletal alteration. The small number of pups and litters resulted in many of these findings occurring at a significantly increased incidence, as compared to the vehicle control group values. These alterations included: short, small or incompletely ossified hyoid ala; a 7th cervical rib; bifid lumbar centrum; misaligned, fused, small or incompletely ossified caudal vertebrae; broad, proximate or short ribs; irregularly shaped manubrium; large sternal centra; irregularly shaped xiphoid or fused metacarpals. The average number of ossification sites per pup per litter was significantly reduced at 1400 mg/kg bw/day for carpals, in comparison to the vehicle control group value. All apparent delays in ossification that were observed in the Caesarean-delivered foetuses at 1400 mg/kg bw/day were resolved by DL21.
The maternal NOAEL was 430 mg/kg/day. The 1400 mg/kg/day dosage caused mortality and reductions in body weight and feed consumption. The developmental NOAEL for pups Caesarean-delivered on day 21 of gestation is 140 mg/kg bw/day. Maternal dosages of 430 and 1400 mg/kg bw/day caused reductions in fetal weight with corresponding delays in fetal skeletal ossification and an increase in the incidence of cervical ribs. All 12 fetuses in the two litters that had live fetuses in the 1400 mg/kg bw/day dosage group had one or more gross, soft tissue and/or skeletal alterations. The 1400 mg/kg bw/day dosage also produced embryo/fetal lethality.
The NOAEL for viability and growth in the offspring naturally delivered and euthanized on day 21 of lactation is 430 mg/kg bw/day. The maternal dosage of 1400 mg/kg bw/day increased the incidence of perinatal mortality. As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, several of the 24 live pups in the two litters that had live pups in the 1400 mg/kg bw/day dosage group on postpartum day 21 had a skeletal alteration. However, all apparent delays in ossification and increased incidences of a cervical rib at the 7th cervical vertebrae that were observed in the Caesareandelivered fetuses in the 430 mg/kg bw/day dosage group were resolved by DL 21.
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]
3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]
4. DATA MATRIX - Reason / purpose for cross-reference:
- read-across: supporting information
- Conclusions:
- In an oral (diet) pre-natal developmental toxicity study in rats, the maternal and developmental NOAELs were 266 mg/kg bw/day, based on transient decrease in maternal feed consumption and body weight gains on GDs 6 and 7 and a slightly higher incidence of delayed ossification in some fetal skeletal elements.
In two dermal (occluded) pre-natal developmental toxicity studies in rats, the maternal and developmental NOAELs were 70 mg/kg bw/day, based on minimal dermal irritation in the dams and small (not statistically significant) reductions in fetal body weights at this dosage level. - Executive summary:
A series of pre-natal developmental toxicity studies (oral: diet, two dermal studies) were described in Politano et al., 2013.
In an pre-natal developmental toxicity study, PEA was administered to 28 female Crl:COBSCD(SD)B rats/group in the diet at dose levels of 0, 1000, 3000, or 10 000 ppm (equivalent to 0, 83, 266 and 799 mg/kg bw/day) from days 6 through 15 of gestation. There were no clinical signs of toxicity or mortality at any dosage level. Feed consumption and body weight gains were reduced predominantly on the first 2 days of treatment at the highest dosage level (results were not statistically different). No other maternal effects were observed during the study. On GD 20, necropsy revealed that 27 of 28 control rats and 26 of 28 rats in each of the low, mid, and -dosage groups were pregnant with live fetuses. Litter parameters, as assessed by mean values for embryo-fetal loss, litter sizes, sex ratios, and litter and mean fetal weights, were equivalent in control and PEA-exposed groups. The incidence, type, and distribution of fetal malformations, anomalies, and skeletal variants were statistically unaffected by maternal exposure to PEA at concentrations as high as 10 000 ppm (799 mg/kg bw/day). However, at the highest dosage level, a marginal delay in fetal ossification might have occurred, as a slightly higher incidence of fetuses with incomplete ossification of some skeletal elements, particularly the sacrocaudal vertebral arches, was observed. The maternal and developmental NOAELs were 266 mg/kg bw/day, transient decrease in maternal feed consumption and body weight gains on GDs 6 and 7 and a slightly higher incidence of delayed ossification in some fetal skeletal elements.
In an pre-natal developmental toxicity study, PEA was administered dermally (occlusive) to 25-35 female Crl:COBSCD(SD)B rats/group at dose levels of 0, 140, 430 and 1400 mg/kg bw/day from days 6 through 15 of gestation. A total of 3 satellite animals in the low- and high-dosage groups were administered radiolabeled PEA on GD 15, after which urine was collected for 24 hours to determine absorption. Local irritation at the application site was observed in some rats early in the treatment period, especially in the high-dosage group. Signs of toxicity included: irritability, hunched posture, walking on toes, pilo erection, and periorbital staining (these effects were restricted almost entirely to dams in the high-dosage group) but resolved by termination. Mean feed consumption and body weight gains were markedly reduced in the high-dosage group during the treatment period. Reversal of these effects occurred upon cessation of treatment, but mean body weights were still statistically depressed at termination. There were no significant differences between control and test groups in mean liver or kidney weights, pregnancy rates, mean preimplantation losses, or gross changes at necropsy on GD 20. Clinical pathology determinations conducted in control and high-dosage dams revealed no adverse hematological or serum chemistry findings. In the absorption study, 63% to 66% of the dosage had been absorbed. Absorption was rapid and extensive, but not dose dependent. In the high-dose group, embryo-fetal toxicity was manifested as statistically significant increases in post-implantation loss with a statistically significant reduction in mean litter size and a marked depression of mean fetal body weight (2.08 g versus 3.26 g in control group, 3.33 g in the 140 mg/kg bw/dayg roup, and 3.18 g in the 430 mg/kg bw/day group). None of these changes occurred at the 2 lower dosages. When fetuses were examined for soft tissue and skeletal abnormalities, a dose-related increase in defects was observed. Statistical analysis of incidences of abnormalities was considered unnecessary given the results obtained. At the highest dosage (1400 mg/kg), morphological changes were present in 160 of the 161 fetuses; the variety, incidence, and degree of the skeletal and soft tissue changes varied considerably among individual fetuses. However, high incidences of the following specific types of defects were noted in more than 40% of the fetuses and 70% of the litters at the high dosage (1400 mg/kg bw/day): anophthalmia or microphthalmia, ventricular septal defects, defects or irregularities affecting thoracic, lumbar, and sacrocaudal vertebrae (associated with short or kinky tail), defects of the thoracic ribs, and occurrence of rudimentary cervical ribs (RCR). At the mid-dosage (430 mg/kg bw/day), occasional fetuses displayed the specific changes observed at 1400 mg/kg bw/day, and the number of fetuses with RCR, thoracic changes, or moderate degrees of reduced ossification exceeded concurrent controls. At the low dosage (140 mg/kg bw/day), findings were equivocal; incidences of RCR and thoracic vertebral irregularities were only marginally higher than those in the controls.
In a corroborative pre-natal developmental toxicity follow up study, PEA was administered dermally (occlusive) to 10 female Crl: COBSCD (SD)BR Charles River rats/group at dose levels of 0, 70, 140, 280, 430 and 700 mg/kg bw/day from days 6 through 15 of gestation. No deaths occurred in the study, but all dosages produced localized, low levels of erythema and/or desquamation at the intrascapular application sites. The severity of these reactions was dose related. Additional signs of toxicity included ptosis and/or urine stained abdominal fur, noted only in dams at the 700 mg/kg bw/day dosage level. Classic signs of maternal toxicity, that is, reduced feed intake and body weight gains, did not occur in a consistent manner, which is often the case when the primary effects are associated with local irritation. Dosages as high as 700 mg/kg bw/day did not adversely affect average litter sizes, numbers of implantations, live fetuses, or post-implantation loss (resorptions). Fetal effects included dose-dependent reductions of the litter mean for live fetal body weights. The reductions were statistically significant at ≥ 140 mg/kg bw/day but not at the lowest dosage of 70 mg/kg bw/day. A significantly increased incidence of RCR occurred at 700 mg/kg bw/day; and minimal increases of reversible delays in ossification were observed at ≥70 mg/kg bw/day. The incidences of the reversible delays in ossification were within the historical ranges of the Testing Facility. Specific delays included: delayed sternal and/or pelvic ossification (all dosage levels); incompletely ossified (hypoplastic) ribs and/or wavy ribs at 70 and at ≥430 mg/kg bw/day; and delays in ossification of the metacarpals and hindpaw phalanges (≥140 mg/kg bw/day), caudal vertebrae (≥280 mg/kg bw/day), forepaw phalanges (≥430 mg/kg bw/day), and sternal centers (700 mg/kg bw/day). The combined results of these two studies indicate maternal and developmental NOAELs of 70 mg/kg bw/day, based on minimal dermal irritation in the dams and small (not statistically significant) reductions in fetal body weights at this dosage level. The decreases in fetal body weights and consequent reversible delays in ossification at 140 mg/kg bw/day and higher were probably associated with maternal stress produced by the localized dermal irritation at the application site. Delays in ossification are considered fetal variations (reversible delays in development) in rodents that are usually related to reduced fetal body weight and, in the absence of fetal malformations, are not considered a significant hazard to humans.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Principles of method if other than guideline:
- This paper describes 3 studies performed with PEA in the 1980s:
1. Oral Developmental Toxicity Study of PEA in Rats (1985)
2. Dermal Developmental Toxicity Study of PEA in Rats (1986)
3. Corroborative Dermal Developmental Toxicity Study of PEA in Rats (1988) - GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:COBSCD(SD)BR (Oral and first dermal study)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River UK Ltd. (Margate, Kent) UK.
- Age at study initiation: Nine-week-old
- Housing: Housed individually in suspended galvanized wire mesh cages
- Diet: Labsure Laboratory Diet No.2 ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 2°C
- Humidity (%): 53% ± 11%
- Photoperiod (hrs dark / hrs light):12-hour light/dark cycle
IN-LIFE DATES: From: To: - Route of administration:
- other: Oral (diet) and dermal (occlusive)
- Details on exposure:
- Oral (diet):
DIET PREPARATION
The PEA was microencapsulated in spray-dried gum arabic and then incorporated into the diet.
First dermal study:
TEST SITE
Phenylethyl alcohol (neat) was applied to a 7 x 5 cm shaved (by electric clipper), interscapular area of the skin
VEHICLE
A control group was administered applications of water at the same mL/kg volume as the high-dosage group (1.40 mL/kg).
USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes. To minimize possible oral ingestion of the control or test material, the dermal applications were occluded with an aluminum foil patch that was held in place by a porous medical adhesive bandage. The applied daily dosages were kept on the animals for 24 hours. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- High-performance liquid chromatographic (HPLC) analyses were used to validate the concentration, homogeneity, and stability of the PEA used throughout the oral (diet) study. No information provided on either dermal study.
- Duration of treatment / exposure:
- DG 6-15 (all studies)
- Frequency of treatment:
- Daily
- Duration of test:
- 20 days
- Dose / conc.:
- 1 000 ppm
- Remarks:
- Equivalent to 83 mg/kg bw/day (oral: diet)
- Dose / conc.:
- 3 000 ppm
- Remarks:
- Equivalent to 266 mg/kg bw/day (oral: diet)
- Dose / conc.:
- 10 000 ppm
- Remarks:
- Equivalent to 799 mg/kg bw/day (oral: diet)
- Dose / conc.:
- 140 mg/kg bw/day (nominal)
- Remarks:
- First dermal study
- Dose / conc.:
- 430 mg/kg bw/day (nominal)
- Remarks:
- First dermal study
- Dose / conc.:
- 1 400 mg/kg bw/day (nominal)
- Remarks:
- First dermal study
- Dose / conc.:
- 70 mg/kg bw/day (nominal)
- Remarks:
- Second dermal study
- Dose / conc.:
- 140 mg/kg bw/day (nominal)
- Remarks:
- Second dermal study
- Dose / conc.:
- 280 mg/kg bw/day (nominal)
- Remarks:
- Second dermal study
- Dose / conc.:
- 430 mg/kg bw/day (nominal)
- Remarks:
- Second dermal study
- Dose / conc.:
- 700 mg/kg bw/day (nominal)
- Remarks:
- Second dermal study
- No. of animals per sex per dose:
- 28 rats/group (oral:diet)
25-35 rats/group (first dermal study)
10 rats/group (second dermal study) - Details on study design:
- Oral (diet):
- Dose selection rationale: The concentrations provided remained unchanged during the course of the study and were selected on the basis of results from a previous pilot study in which dietary inclusion of up to 9000 ppm PEA was well tolerated by pregnant rats and appeared to maintain diet palatability.
First dermal study:
- Dose selection rationale: The 430 mg/kg bw/day dosage was selected because it was approximately equivalent to the oral high dosage used in the Mankes et al., 1983 study. The highest dosage, 1400 mg/kg bw/day, was used to extend the range of dosages tested, in case there was differential absorption by the dermal route.
Second dermal study:
- Dose selection rationale: The object of this second topical study was to corroborate the finding of dose-dependent increases in fetal and litter incidences of cervical ribs at 140, 430, and 1400 mg/kg bw/day found in the first topical study and to define more accurately the dosages that may have produced the reported fetal changes. - Maternal examinations:
- See below
- Ovaries and uterine content:
- See below
- Fetal examinations:
- See below
- Statistics:
- Statistical analyses of clinical observation and other proportion data were performed using the variance test for homogeneity of the binomial distribution.23 Continuous data (eg, body weights and body weight changes) were analyzed using Bartlett test of homogeneity of variances and the analysis of variance (ANOVA), when appropriate (ie, Bartlett test was not significant, P > .001). If the ANOVA was significant (P ≤ .05), Dunnett test was used to identify the statistical significance of the individual groups. If the ANOVA was not appropriate (ie, Bartlett’s test was significant, P ≤ .001), the Kruskal-Wallis test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis test was statistically significant (P≤.05), Dunn method of multiple comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher exact test was used to analyze the data.
- Dose descriptor:
- NOAEL
- Remarks:
- Oral (diet)
- Effect level:
- 266 mg/kg bw/day (nominal)
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- Dose descriptor:
- NOAEL
- Remarks:
- Dermal studies
- Effect level:
- 70 mg/kg bw/day (nominal)
- Basis for effect level:
- dermal irritation
- Abnormalities:
- no effects observed
- Dose descriptor:
- NOAEL
- Remarks:
- Oral (diet)
- Effect level:
- 266 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- skeletal malformations
- Dose descriptor:
- NOAEL
- Remarks:
- Dermal studies
- Effect level:
- 70 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- fetal/pup body weight changes
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- skeletal: vertebra
- Description (incidence and severity):
- At 799 mg/kg bw/day in the oral (diet) study, a slightly higher incidence of fetuses with incomplete ossification of some skeletal elements, particularly the sacrocaudal vertebral arches, was observed.
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 70 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- not specified
- Relevant for humans:
- no
- Conclusions:
- In an oral (diet) pre-natal developmental toxicity study in rats, the maternal and developmental NOAELs were 266 mg/kg bw/day, based on transient decrease in maternal feed consumption and body weight gains on GDs 6 and 7 and a slightly higher incidence of delayed ossification in some fetal skeletal elements.
In two dermal (occluded) pre-natal developmental toxicity studies in rats, the maternal and developmental NOAELs were 70 mg/kg bw/day, based on minimal dermal irritation in the dams and small (not statistically significant) reductions in fetal body weights at this dosage level. - Executive summary:
A series of pre-natal developmental toxicity studies (oral: diet, two dermal studies) were described in Politano et al., 2013.
In an pre-natal developmental toxicity study, PEA was administered to 28 female Crl:COBSCD(SD)B rats/group in the diet at dose levels of 0, 1000, 3000, or 10 000 ppm (equivalent to 0, 83, 266 and 799 mg/kg bw/day) from days 6 through 15 of gestation. There were no clinical signs of toxicity or mortality at any dosage level. Feed consumption and body weight gains were reduced predominantly on the first 2 days of treatment at the highest dosage level (results were not statistically different). No other maternal effects were observed during the study. On GD 20, necropsy revealed that 27 of 28 control rats and 26 of 28 rats in each of the low, mid, and -dosage groups were pregnant with live fetuses. Litter parameters, as assessed by mean values for embryo-fetal loss, litter sizes, sex ratios, and litter and mean fetal weights, were equivalent in control and PEA-exposed groups. The incidence, type, and distribution of fetal malformations, anomalies, and skeletal variants were statistically unaffected by maternal exposure to PEA at concentrations as high as 10 000 ppm (799 mg/kg bw/day). However, at the highest dosage level, a marginal delay in fetal ossification might have occurred, as a slightly higher incidence of fetuses with incomplete ossification of some skeletal elements, particularly the sacrocaudal vertebral arches, was observed. The maternal and developmental NOAELs were 266 mg/kg bw/day, based on the transient decrease in maternal feed consumption and body weight gains on GDs 6 and 7 and a slightly higher incidence of delayed ossification in some fetal skeletal elements.
In an pre-natal developmental toxicity study, PEA was administered dermally (occlusive) to 25-35 female Crl:COBSCD(SD)B rats/group at dose levels of 0, 140, 430 and 1400 mg/kg bw/day from days 6 through 15 of gestation. A total of 3 satellite animals in the low- and high-dosage groups were administered radiolabeled PEA on GD 15, after which urine was collected for 24 hours to determine absorption. Local irritation at the application site was observed in some rats early in the treatment period, especially in the high-dosage group. Signs of toxicity included: irritability, hunched posture, walking on toes, pilo erection, and periorbital staining (these effects were restricted almost entirely to dams in the high-dosage group) but resolved by termination. Mean feed consumption and body weight gains were markedly reduced in the high-dosage group during the treatment period. Reversal of these effects occurred upon cessation of treatment, but mean body weights were still statistically depressed at termination. On GD 20, necropsy revealed that 21 of 25 control rats and 30, 22 and 18 rats in each of the low, mid, and -dosage groups were pregnant with live fetuses. There were no significant differences between control and test groups in mean liver or kidney weights, pregnancy rates, mean preimplantation losses, or gross changes at necropsy on GD 20. Clinical pathology determinations conducted in control and high-dosage dams revealed no adverse hematological or serum chemistry findings. In the absorption study, 63% to 66% of the dosage had been absorbed. Absorption was rapid and extensive, but not dose dependent. In the high-dose group, embryo-fetal toxicity was manifested as statistically significant increases in post-implantation loss with a statistically significant reduction in mean litter size and a marked depression of mean fetal body weight (2.08 g versus 3.26 g in control group, 3.33 g in the 140 mg/kg bw/dayg roup, and 3.18 g in the 430 mg/kg bw/day group). None of these changes occurred at the 2 lower dosages. When fetuses were examined for soft tissue and skeletal abnormalities, a dose-related increase in defects was observed. Statistical analysis of incidences of abnormalities was considered unnecessary given the results obtained. At the highest dosage (1400 mg/kg), morphological changes were present in 160 of the 161 fetuses; the variety, incidence, and degree of the skeletal and soft tissue changes varied considerably among individual fetuses. However, high incidences of the following specific types of defects were noted in more than 40% of the fetuses and 70% of the litters at the high dosage (1400 mg/kg bw/day): anophthalmia or microphthalmia, ventricular septal defects, defects or irregularities affecting thoracic, lumbar, and sacrocaudal vertebrae (associated with short or kinky tail), defects of the thoracic ribs, and occurrence of rudimentary cervical ribs (RCR). At the mid-dosage (430 mg/kg bw/day), occasional fetuses displayed the specific changes observed at 1400 mg/kg bw/day, and the number of fetuses with RCR, thoracic changes, or moderate degrees of reduced ossification exceeded concurrent controls. At the low dosage (140 mg/kg bw/day), findings were equivocal; incidences of RCR and thoracic vertebral irregularities were only marginally higher than those in the controls.
In a corroborative pre-natal developmental toxicity follow up study, PEA was administered dermally (occlusive) to 10 female Crl: COBSCD (SD)BR Charles River rats/group at dose levels of 0, 70, 140, 280, 430 and 700 mg/kg bw/day from days 6 through 15 of gestation. No deaths occurred in the study, but all dosages produced localized, low levels of erythema and/or desquamation at the intrascapular application sites. The severity of these reactions was dose related. Additional signs of toxicity included ptosis and/or urine stained abdominal fur, noted only in dams at the 700 mg/kg bw/day dosage level. Classic signs of maternal toxicity, that is, reduced feed intake and body weight gains, did not occur in a consistent manner, which is often the case when the primary effects are associated with local irritation. Dosages as high as 700 mg/kg bw/day did not adversely affect average litter sizes, numbers of implantations, live fetuses, or post-implantation loss (resorptions). Fetal effects included dose-dependent reductions of the litter mean for live fetal body weights. The reductions were statistically significant at ≥ 140 mg/kg bw/day but not at the lowest dosage of 70 mg/kg bw/day. A significantly increased incidence of RCR occurred at 700 mg/kg bw/day; and minimal increases of reversible delays in ossification were observed at ≥70 mg/kg bw/day. The incidences of the reversible delays in ossification were within the historical ranges of the Testing Facility. Specific delays included: delayed sternal and/or pelvic ossification (all dosage levels); incompletely ossified (hypoplastic) ribs and/or wavy ribs at 70 and at ≥430 mg/kg bw/day; and delays in ossification of the metacarpals and hindpaw phalanges (≥140 mg/kg bw/day), caudal vertebrae (≥280 mg/kg bw/day), forepaw phalanges (≥430 mg/kg bw/day), and sternal centers (700 mg/kg bw/day). The combined results of these two studies indicate maternal and developmental NOAELs of 70 mg/kg bw/day, based on minimal dermal irritation in the dams and small (not statistically significant) reductions in fetal body weights at this dosage level. The decreases in fetal body weights and consequent reversible delays in ossification at 140 mg/kg bw/day and higher were probably associated with maternal stress produced by the localized dermal irritation at the application site. Delays in ossification are considered fetal variations (reversible delays in development) in rodents that are usually related to reduced fetal body weight and, in the absence of fetal malformations, are not considered a significant hazard to humans.
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- disregarded due to major methodological deficiencies
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]
3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]
4. DATA MATRIX - Reason / purpose for cross-reference:
- read-across: supporting information
- Dose descriptor:
- other: No NOEL or NOAEL identified
- Abnormalities:
- not specified
- Dose descriptor:
- other: No NOEL or NOAEL identified
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 0 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- In a disregarded pre-natal developmental toxicity study in Long-Evans rats, no NOEL or NOAEL was identifed. Examination of offspring revealed adverse reproductive and teratogenic effects in a dose-related manner.
- Executive summary:
In a disregarded developmental toxicity study (Mankes et al., 1983) PEA was administered to 5-7 female Long Evans rats by gavage at dose levels of 0, 4.3, 43 and 432 mg/kg bw/day from days 6 through 15 of gestation.
Signs of severe maternal intoxication were noted immediately following ingestion at 432 mg/kg bw/day. These signs persisted overnight and were observed after each daily dose. Lower levels (43 or 4.3 mg/kg bw/day were asymptomatic. One dam (432 mg/kg bw/day) died from intratracheal intubation.
All offspring (100%) at 432 mg/kg bw/day were either dead or malformed. Lower levels caused fewer adverse effects in the offspring (97% at 43 mg/kg bw/day, 55% at 4.3 mg/kg bw/day). Intrauterine growth retardation occurred at 432 mg/kg bw/day and at 4.3 mg/kg bw/day. Average pup weight and crown-rump length were decreased (pup weight from 3.6 g in controls to 2.5 g at 432 mg/kg bw/day and 3.1 g at 4.3 mg/kg bw/day; and length from 3.7 cm in controls to 3.2 cm at 432 mg/kg bw/day and 3.5 cm at 4.3 mg/kg bw/day) on a per litter basis. Increased numbers (32 at 432 mg/kg bw/day and 5 at 4.3 mg/kg bw/day) of grossly runted (weight less than 2.7 g) live offspring were also noted in these groups. Embryolethality did not occur at 432 mg/kg bw/day. Mean litter size was decreased to 9 pups in at 43 mg/kg bw/day and at 4.3 mg/kg bw/day, as compared to 12 pups in control litters. This difference was not a significant decrease at 43 mg/kg bw/day due to the large (standard deviation = 7) variability. The 43 mg/kg bw/day fetal death rate (total of resorptions and stillborn) was increased significantly to 18% (compared to 6% in controls).
A clear dose-related increase in malformations was caused by 2-phenylethanol. At the lowest (4.3 mg/kg bw/day) exposure group, 30 of 60 live births in all 7 litters were malformed; at 43 mg/kg bw/day, 93% were malformed from 7 of 7 litters; and at the highest exposure group (432 mg/kg bw/day), all 51 live offspring from 4 of 4 litters were grossly malformed.
At 432 mg/kg bw/day, pups had malformed eyes (31 of 51), malformed limbs (21 of 51), hydronephrosis (15 of 51), neural-tube defects (12 of 51) and malformed digits (6 of 51). Figure 1 shows a grossly malformed pup at 432 mg/kg bw/day; note the micromelia, club and web foot, microphthalmia, and open eyes. Offspring from 43 mg/kg bw/day had malformed eyes (28 of 65), hydronephrosis (19 of 65), and limb defects (3 of 65). Eye defects (9 of 60) and hydronephrosis (5 of 60) occurred in 4.3 mg/kg bw/day pups.
Reduced cranial-bone ossification occurred in all groups. Limb ossification was markedly reduced in micromelic pups at 432 mg/kg bw/day and at 43 mg/kg bw/day. The radius, ulna, and humerus appear to be extremely short with no cartilage. Other bony defects included an overall reduction in size (micrognathia), and lack of pelvis, tail bones, carpals, or tarsals. Variant (reduced) ossification of the rib and tail were noted in 16 43 mg/kg bw/day pups. Sternebral variations were noted in 5 of 60 4.3 mg/kg bw/day pups.
This pre-natal development toxicity study in the rat is not acceptable and does not satisfy the guideline requirement for this oral study (OECD 414) in rats. The number of pregnant rats were 5-7 per group, it was not conducted according to GLP and many maternal parameters are not described in the publication.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- disregarded due to major methodological deficiencies
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- significant methodological deficiencies
- Remarks:
- The number of pregnant rats were 5-7 per group, it was not conducted according to GLP and many maternal parameters are not described in the publication.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- GLP compliance:
- no
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Fisher Chemicals Co; Lot 795884 - Species:
- rat
- Strain:
- Long-Evans
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Blue Spruce Farms in Altamont, N.Y. USA
- Weight at study initiation: 200-300 g - Route of administration:
- oral: gavage
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: Aqueous suspensions of PEA were freshly prepared, and daily doses of 20 ml/kg were given by gavage based on their current body weights.
- Analytical verification of doses or concentrations:
- no
- Details on mating procedure:
- The presence of vaginal sperm was defined as d 0 of gestation. Rats that did not mate and those that did not become pregnant were excluded
rom the analysis. - Duration of treatment / exposure:
- DG 6 - 15
- Frequency of treatment:
- Daily
- Duration of test:
- 20 days
- Dose / conc.:
- 4.3 mg/kg bw/day (nominal)
- Dose / conc.:
- 43 mg/kg bw/day (nominal)
- Dose / conc.:
- 432 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 4.3 mg/kg bw/day: 7 females
43 mg/kg bw/day: 7 females
432 mg/kg bw/day: 5 females - Control animals:
- yes
- Maternal examinations:
- DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Dams were observed daily for signs of toxicity and lethality.
BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were recorded on d 0-15 and 20 of gestation.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: All dams were overdosed with ether on DG 20 and their fetuses were delivered by Caesarean section. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included: The following observations and weights were recorded at DG 20: total uterine weight, total litter weight, individual pup weights, number of live pups, stillbirths, résorptions, implantation sites, sex distribution, crown-rump length, and number of corpora lutea. - Fetal examinations:
- All live pups were examined for gross malformations at birth: soft-tissue (internal) defects were evaluated by the method of Wilson while skeletal variations were detected by the method of McLeod.
- Statistics:
- The t-squared (BMDP3D) analysis was used to compare uterine weights, litter weights, pup weights, litter sizes and crown-rump length. The two-way frequency count routine (BMDP1F) was applied to embryonic deaths (resorptions), total of dead and malformed, and incidences of malformations.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Signs of severe maternal intoxication were noted immediately following ingestion at 432 mg/kg bw/day. These signs persisted overnight and were observed after each daily dose. Lower levels (43 or 4.3 mg/kg bw/day were asymptomatic.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One dam (432 mg/kg bw/day) died from intratracheal intubation.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Reduced weight gain was evident at the 43 mg/kg dos
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- 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:
- not specified
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not examined
- Number of abortions:
- not specified
- Pre- and post-implantation loss:
- not specified
- Total litter losses by resorption:
- effects observed, treatment-related
- Description (incidence and severity):
- The 43 mg/kg bw/day fetal death rate (total of resorptions and stillborn) was increased significantly to 18% (compared to 6% in controls).
- Early or late resorptions:
- not specified
- Dead fetuses:
- effects observed, treatment-related
- Description (incidence and severity):
- All offspring (100%) at 432 mg/kg bw/day were either dead or malformed. Lower levels caused fewer adverse effects in the offspring (97% at 43 mg/kg bw/day, 55% at 4.3 mg/kg bw/day; Table 1).
- Changes in pregnancy duration:
- not specified
- Changes in number of pregnant:
- not specified
- Dose descriptor:
- other: No NOEL or NOAEL identified
- Abnormalities:
- not specified
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Intrauterine growth retardation occurred at 432 mg/kg bw/day and at 4.3 mg/kg bw/day. Average pup weight and crown-rump length were decreased (pup weight from 3.6 g in controls to 2.5 g at 432 mg/kg bw/day and 3.1 g at 4.3 mg/kg bw/day; and length from 3.7 cm in controls to 3.2 cm at 432 mg/kg bw/day and 3.5 cm at 4.3 mg/kg bw/day) on a per litter basis (Table 1).
- Reduction in number of live offspring:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased numbers (32 at 432 mg/kg bw/day and 5 at 4.3 mg/kg bw/day) of grossly runted (weight less than 2.7 g) live offspring were also noted in these groups (Table 2). Embryolethality did not occur at 432 mg/kg bw/day.
- Changes in sex ratio:
- not specified
- Changes in litter size and weights:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean litter size was decreased to 9 pups in at 43 mg/kg bw/day and at 4.3 mg/kg bw/day, as compared to 12 pups in control litters. This difference was not a significant decrease at 43 mg/kg bw/day due to the large (standard deviation = 7) variability (Table 1).
- Changes in postnatal survival:
- not specified
- External malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- A clear dose-related increase in malformations was caused by 2-phenylethanol (Table 2). At the lowest (4.3 mg/kg bw/day) exposure group, 30 of 60 live births in all 7 litters were malformed; at 43 mg/kg bw/day , 93% were malformed from 7 of 7 litters; and at the highest exposure group (432 mg/kg bw/day), all 51 live offspring from 4 of 4 litters were grossly malformed.
- Skeletal malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- Reduced cranial-bone ossification occurred in all groups (Table 2). As seen in Figs. 2 and 3, the bones of the skull are poorly ossified. Limb ossification was markedly reduced in micromelic pups at 432 mg/kg bw/day and at 43 mg/kg bw/day. The radius, ulna, and humerus appear to be extremely short with no cartilage (Fig. 2). Other bony defects included an overall reduction in size (micrognathia), and lack of pelvis, tail bones, carpals, or tarsals. Illustrated in Fig. 3 is evidence of a clear neural-tube defect in the neck region. Other defects noted were in the rib, pelvis and cranium (Fig. 3). A well developed skeleton of a normal control is presented in Fig. 4 for comparative purposes. Variant (reduced) ossification of the rib and tail were noted in 16 43 mg/kg bw/day pups. Sternebral variations were noted in 5 of 60 4.3 mg/kg bw/day pups (Table 2).
- Visceral malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- At 432 mg/kg bw/day, pups had malformed eyes (31 of 51), malformed limbs (21 of 51), hydronephrosis (15 of 51), neural-tube defects (12 of 51) and malformed digits (6 of 51). Figure 1 shows a grossly malformed pup at 432 mg/kg bw/day; note the micromelia, club and web foot, microphthalmia, and open eyes. Offspring from 43 mg/kg bw/day had malformed eyes (28 of 65), hydronephrosis (19 of 65), and limb defects (3 of 65). Eye defects (9 of 60) and hydronephrosis (5 of 60) occurred in 4.3 mg/kg bw/day pups (Table 2).
- Dose descriptor:
- other: No NOEL or NOAEL identified
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 0 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- In a disregarded pre-natal developmental toxicity study in Long-Evans rats, no NOEL or NOAEL was identifed. Examination of offspring revealed adverse reproductive and teratogenic effects in a dose-related manner.
- Executive summary:
In a disregarded developmental toxicity study (Mankes et al., 1983) PEA was administered to 5-7 female Long Evans rats by gavage at dose levels of 0, 4.3, 43 and 432 mg/kg bw/day from days 6 through 15 of gestation.
Signs of severe maternal intoxication were noted immediately following ingestion at 432 mg/kg bw/day. These signs persisted overnight and were observed after each daily dose. Lower levels (43 or 4.3 mg/kg bw/day were asymptomatic. One dam (432 mg/kg bw/day) died from intratracheal intubation.
All offspring (100%) at 432 mg/kg bw/day were either dead or malformed. Lower levels caused fewer adverse effects in the offspring (97% at 43 mg/kg bw/day, 55% at 4.3 mg/kg bw/day). Intrauterine growth retardation occurred at 432 mg/kg bw/day and at 4.3 mg/kg bw/day. Average pup weight and crown-rump length were decreased (pup weight from 3.6 g in controls to 2.5 g at 432 mg/kg bw/day and 3.1 g at 4.3 mg/kg bw/day; and length from 3.7 cm in controls to 3.2 cm at 432 mg/kg bw/day and 3.5 cm at 4.3 mg/kg bw/day) on a per litter basis. Increased numbers (32 at 432 mg/kg bw/day and 5 at 4.3 mg/kg bw/day) of grossly runted (weight less than 2.7 g) live offspring were also noted in these groups. Embryolethality did not occur at 432 mg/kg bw/day. Mean litter size was decreased to 9 pups in at 43 mg/kg bw/day and at 4.3 mg/kg bw/day, as compared to 12 pups in control litters. This difference was not a significant decrease at 43 mg/kg bw/day due to the large (standard deviation = 7) variability. The 43 mg/kg bw/day fetal death rate (total of resorptions and stillborn) was increased significantly to 18% (compared to 6% in controls).
A clear dose-related increase in malformations was caused by 2-phenylethanol. At the lowest (4.3 mg/kg bw/day) exposure group, 30 of 60 live births in all 7 litters were malformed; at 43 mg/kg bw/day, 93% were malformed from 7 of 7 litters; and at the highest exposure group (432 mg/kg bw/day), all 51 live offspring from 4 of 4 litters were grossly malformed.
At 432 mg/kg bw/day, pups had malformed eyes (31 of 51), malformed limbs (21 of 51), hydronephrosis (15 of 51), neural-tube defects (12 of 51) and malformed digits (6 of 51). Figure 1 shows a grossly malformed pup at 432 mg/kg bw/day; note the micromelia, club and web foot, microphthalmia, and open eyes. Offspring from 43 mg/kg bw/day had malformed eyes (28 of 65), hydronephrosis (19 of 65), and limb defects (3 of 65). Eye defects (9 of 60) and hydronephrosis (5 of 60) occurred in 4.3 mg/kg bw/day pups.
Reduced cranial-bone ossification occurred in all groups. Limb ossification was markedly reduced in micromelic pups at 432 mg/kg bw/day and at 43 mg/kg bw/day. The radius, ulna, and humerus appear to be extremely short with no cartilage. Other bony defects included an overall reduction in size (micrognathia), and lack of pelvis, tail bones, carpals, or tarsals. Variant (reduced) ossification of the rib and tail were noted in 16 43 mg/kg bw/day pups. Sternebral variations were noted in 5 of 60 4.3 mg/kg bw/day pups.
This pre-natal development toxicity study in the rat is not acceptable and does not satisfy the guideline requirement for this oral study (OECD 414) in rats. The number of pregnant rats were 5-7 per group, it was not conducted according to GLP and many maternal parameters are not described in the publication.
Referenceopen allclose all
Rats Assigned To Natural Delivery Observations
Mortality
One rat in the 1400 mg/kg bw/day dosage group was found dead on DG 13. This death was presumed related to treatment with Phenethyl Alcohol because: 1) similar events occurred in the rats assigned to Caesarean-sectioning; and 2) the observation occurred in the high dosage group. In-life and postmortem observations in this rat are further summarized in the following text. All other rats assigned to the natural delivery phase of this study survived to scheduled sacrifice. Rat 7267 was administered the test article seven times. This rat had skin observations of flaking grade 2 and erythema grade 2 on DG 13, and clinical signs of ptosis, decreased motor activity, vocalization during rinsing, red perivaginal substance and mild dehydration (based on skin turgor) on the day of or during days prior to death. This rat lost 25 g of body weight from DG 12 to DG 13, and it consumed 11 g of feed during this time period. Necropsy revealed a mottled (red and dark red) appearance to all lobes of the lungs. This rat was pregnant.
Skin Reactions
Observations consistent with primary irritancy occurred in female rats percutaneously administered Phenethyl Alcohol during the gestation period (DGs 7 through 21). The onset and severity of the skin reactions was, in general, dependent on the dosage of Phenethyl Alcohol. Erythema grade 2 (distinct redness) and edema grade 2 (moderate, raised 1 mm to 2 mm) occurred in more rats (significant at p≤ 0.01) in the 1400 mg/kg bw/day dosage group, in comparison to the vehicle control group values. Two rats in the 1400 mg/kg bw/day dosage group also had an observation of erythema grade 3 (“beet red” color) on one or three occasions during the dosage period; no other rat had this skin reaction. Significantly more (p≤ 0.01) rats in the 140, 430 and 1400 mg/kg bw/day dosage groups had flaking grade 1 (barely perceptible scales) than rats in the corresponding vehicle control group. In addition, 8, 15 and 20 rats in the 140, 430 and 1400 mg/kg bw/day dosage groups, respectively, had flaking grade 2 (distinct scales). The number of rats with this observation was significant (p≤ 0.01) at 430 and 1400 mg/kg bw/day, as compared to the vehicle control group value. Significantly more (p≤ 0.01) rats in the 1400 mg/kg bw/day dosage group had flaking grade 3 (pronounced flaking with denuded sites) than rats in the corresponding vehicle control group. All other skin reactions occurred at a comparable incidence relative to the vehicle control group value and did not differ significantly. Erythema grade 1 (barely perceptible redness) and scabbing was observed in each dosage group, including the vehicle control group. One rat in the 1400 mg/kg bw/day dosage group had edema grade 1 (mild, raised <1 mm) on two occasions.
Clinical Observations
Clinical signs attributed to treatment with Phenethyl Alcohol that achieved statistical significance are summarized in Text Table 3. The number of affected rats in the 1400 mg/kg bw/day dosage group during the gestation period was significantly increased (p≤ 0.01) in comparison to the vehicle control group values. In addition, low incidences of impaired righting reflex, chromorhinorrhea, cold to touch, red substance in the cage pan, hyperreactivity to touch, black perinasal substance, mild to moderate dehydration (based on skin turgor) and red substance on the fur occurred in the 1400 mg/kg bw/day dosage group during the dosage period. The occurrence of these clinical signs did not differ significantly from the vehicle control group value (p> 0.05). However, these observations were also attributed to treatment with Phenethyl Alcohol because the clinical signs occurred in rats that had concomitant signs of clinical toxicity. All other clinical signs observed during the gestation period were considered unrelated to percutaneous administration of Phenethyl Alcohol because: 1) the incidences were not dosage-dependent; and 2) the number of affected rats in a group was not considerably greater than the number affected in the vehicle control group. These clinical observations included incisors misaligned, excess salivation, sparse hair coat (underside), swollen right ear, neck and head and a mass on the chest. In addition, observations such as urinestained abdominal fur, red or brown perivaginal substance, swollen right ear and red perianal substance that occurred in one or two rats during the lactation period were considered unrelated to treatment with Phenethyl Alcohol because these signs were not dosage-dependent. (Summaries - Tables B1 and B17)
Maternal Body Weights and Body Weight Changes
Gestation
Body weight losses occurred in each dosage group, including the vehicle control group, during the first three days of the dosage period (DGs 7 to 10). At 1400 mg/kg bw/day, the body weight losses reached statistical significance relative to the vehicle control group value (p≤0.01), and continued to persist on DGs 10 to 14 (significant at p≤0.01). Thereafter, body weight gains were reduced or significantly reduced (p≤0.01) in the 1400 mg/kg bw/day dosage group, in comparison to the vehicle control group value (62% and 9% lower than vehicle control value on DGs 14 to 17 and DGs 17 to 21, respectively). Overall, body weight gains were significantly reduced (p≤0.01) for the entire gestation dosage period (DGs 7 to 21) and the entire gestation period (DGs 0 to 21), as compared to the vehicle control group values. Body weight gains in the 140, 430 and 1400 mg/kg bw/day dosage groups were 99%, 95% and 38% of the vehicle control group value, respectively, on DGs 7 to 21. Reflecting the effects of Phenethyl Alcohol on body weight gains, the average maternal body weight in the 1400 mg/kg bw/day dosage groups was significantly reduced (p≤0.05or p≤0.01) beginning on DG 8 and continuing until DG 21. The average maternal body weight on DG 21 was 99%, 98% and 82% of the vehicle control group value in the 140, 430 and 1400 mg/kg bw/day dosage groups, respectively. Body weights and body weight gains during the gestation period were unaffected by dosages of Phenethyl Alcohol as high as 430 mg/kg bw/day.
Lactation
Body weight gains were significantly reduced (p≤0.05) in the 430 and 1400 mg/kg bw/day dosage groups on Days 1 to 4 of lactation (DLs 1 to 4). Thereafter, body weight gains were comparable among the dosage groups. Body weight gains in the 140, 430 and 1400 mg/kg bw/day dosage groups were 97%, 86% and 96% of the vehicle control group value, respectively, on DLs 1 to 21. Body weights and body weight gains during the lactation period were unaffected by dosages of Phenethyl Alcohol as high as 140 mg/kg bw/day. The average maternal body weight on DL 21 was 100%, 98% and 104% of the vehicle control group value in the 140, 430 and 1400 mg/kg bw/day dosage groups, respectively. (Figure 2; Summaries - Tables B2 through B5)
Maternal Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values
Gestation
Absolute feed consumption values were reduced or significantly reduced (p≤0.01) in the 1400 mg/kg bw/day dosage group for the entire dosage period (calculated as DGs 7 to 21) and at all tabulated intervals within the dosage period, in comparison to the vehicle control group values. Absolute feed consumption values in the 140, 430 and 1400 mg/kg bw/day dosage groups were 99%, 95% and 82% of the vehicle control group value, respectively, for the entire dosage period (calculated as DGs 7 to 21). Relative to body weight, gestating rats in the 1400 mg/kg bw/day dosage group consumed less or significantly less (p≤0.01) feed on DGs 7 to 10, DGs 10 to 14 and DGs 14 to 17, in comparison to the vehicle control group values. Overall, relative feed consumption in the 1400 mg/kg bw/day was reduced by 6% for the entire dosage period (calculated as DGs 7 to 21), as compared to the vehicle control group value. Absolute and relative feed consumption values during the gestation period were unaffected by dosages of Phenethyl Alcohol as high as 430 mg/kg bw/day.
Lactation
Corresponding to reductions in body weight gain, absolute and relative feed consumption values were reduced in the 1400 mg/kg bw/day dosage group on DLs 1 to 4 and overall for DLs 1 to 14, in comparison to the vehicle control group values (12% and 10% lower than the vehicle control group values, respectively). Relative to body weights, lactating dams consumed less feed (significant at p≤0.05 or p≤0.01) on DLs 4 to 7, DLs 7 to 10 and overall for DLs 1 to 14, in comparison to the vehicle control group values. Absolute and relative feed consumption values during the lactation period were unaffected by dosages of Phenethyl Alcohol as high as 430 mg/kg bw/day. The significant reduction (p≤0.05) in absolute feed consumption that occurred on DLs 7 to 10 in the 430 mg/kg bw/day dosage group was considered unrelated to treatment with Phenethyl Alcohol because the reduction was transient and did not persist. (Summaries - Tables B6 through B9)
Natural Delivery Observations
Pregnancy occurred in 20, 20, 19 and 16 of the 25 mated female rats in the 0 (Vehicle Control), 140, 430 and 1400 mg/kg bw/day dosage groups, respectively. All pregnant dams in the 140 and 430 mg/kg bw/day dosage groups delivered litters. At 1400 mg/kg bw/day, 8 of the 16 pregnant rats (p≤0.01) delivered a litter. Of these 8 dams, one had a litter with no liveborn pups and four had all pups die before day 4 postpartum. One of the 8 dams was in the process of delivering, but on the following day no pups were accounted for (i.e., all pups were presumed cannibalized). Seven of the 16 dams did not deliver a litter by DG 25 and were subsequently euthanized, and another dam was found dead on DG 13, as previously described. Only two dams (no. 7265 and 7279) in the 1400 mg/kg bw/day dosage group had viable pups that survived until day 21 postpartum. The duration of gestation was significantly increased (p≤0.01) in the 1400 mg/kg bw/day dosage group (24.0 days vs. 23.0 days in vehicle controls). In addition, the gestation index (number of dams with one or more liveborn pups/number of pregnant rats) at 1400 mg/kg bw/day was significantly reduced (p≤0.01), as compared to the vehicle control group value (40% vs. 100% in vehicle controls). The averages for the total number of pups delivered and the number of liveborn pups were reduced or significantly reduced (p≤0.01) in the 1400 mg/kg bw/day dosage group, in comparison to the vehicle control group values. The percentage of pups that died or were presumed cannibalized on day 1 and days 2 to 4 postpartum was significantly increased (p_ 0.01) in the 1400 mg/kg bw/day dosage group, as compared to the vehicle control group values. Reflecting this increase in pup mortality, the viability index at 1400 mg/kg bw/day was significantly reduced (p_ 0.01) relative to the vehicle control group value (63.2% vs. 99.0% in vehicle controls). The average number of surviving pups per litter was significantly reduced (p≤0.01) in the 1400 mg/kg bw/day dosage group on days 4, 7, 14 and 21 postpartum, in comparison to the vehicle control group values. In addition, the average pup weight per litter was significantly lower (p≤0.01) in the 1400 mg/kg bw/day dosage group on day 1 postpartum relative to the vehicle control group value (16% lower than the vehicle control group value). On postpartum days 4, 7 and 21, pup body weights at 1400 mg/kg bw/day were 9%, 10% and 7% lower than the corresponding vehicle control group values. Natural delivery and litter observations were unaffected by dosages of Phenethyl Alcohol as high as 430 mg/kg bw/day. Values for the average number of implantation sites per delivered litter, the number of dams with stillborn pups, lactation index, percent male pups per number of pups sexed per litter and live litter size at weighing were comparable among the four dosage groups and did not significantly differ. (Summaries - Tables B10 and B11)
Clinical and Necropsy Observations - F1 Generation Pups
At 1400 mg/kg bw/day, a significant number of litters (p≤0.01) had pups with mild dehydration (based on skin turgor), a thread-like tail and were not nursing. No clinical observations in the F1 generation pups were attributed to dosages of Phenethyl Alcohol as high as 430 mg/kg bw/day because: 1) the incidences were not dosage dependent; 2) the observation occurred in only one litter; and/or 3) the observation occurred only in the vehicle control group. These clinical observations included bent tail, pale appearance, a fissure on the left hindlimb, gasping, cold to touch, no milk band present, a scab along the lower midline, ungroomed coat and a red appearance to the lower midline. Reflecting the number of pups that survived to scheduled sacrifice, fewer pups (litter and pup incidence significant at p≤0.01) from the 1400 mg/kg bw/day dosage group appeared normal at necropsy. One pup in the 1400 mg/kg bw/day dosage group that survived to scheduled sacrifice had extreme dilation of the pelvis in the left kidney. The toxicological significance of this finding could not be determined because it occurred in one pup from one litter in the 1400 mg/kg bw/day dosage group. No other gross lesions occurred in pups that survived to scheduled sacrifice. No milk in the stomach occurred in 1, 1, 2 and 4 pups that were found dead in the 0 (Vehicle Control), 140, 430 and 1400 mg/kg bw/day dosage groups, respectively. (Summaries - Tables B12 and B13).
Pup Alterations
Pup alterations were defined as: 1) malformations (irreversible changes that occur at low incidences in this species and strain); or 2) variations (common findings in this species and strain and reversible delays or accelerations in development). Litter averages were calculated for specific pup ossification sites as part of the evaluation of the degree of pup ossification. Pup evaluations on DL 21 were based on 282, 276, 249 and 24 live delivered pups in 20, 20, 19 and 2 litters in the 0 (Vehicle Control), 140, 430 and 1400 mg/kg bw/day dosage groups, respectively. Each of these pups was examined for skeletal external alterations and ossification site averages. As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, several of the 24 live pups in the two litters that had live pups in the 1400 mg/kg bw/day dosage group on postpartum day 21 had a skeletal alteration. The small number of pups and litters resulted in many of these findings occurring at a significantly increased (p≤0.01) incidence compared to the vehicle control group values. These alterations included: short, small or incompletely ossified hyoid ala; a 7th cervical rib; bifid lumbar centrum; short ribs; irregularly shaped manubrium; large sternal centra; irregularly shaped xiphoid or fused metacarpals. The average number of ossification sites per pup per litter was significantly reduced (p≤0.01) in the 1400 mg/kg bw/day dosage group for carpals, in comparison to the vehicle control group value. No skeletal pup malformations occurred and no variations were caused by dosages of Phenethyl Alcohol as high as 430 mg/kg bw/day. Pup ossification site averages were comparable among the 0 (Vehicle Control), 140 and 430 mg/kg bw/day dosage groups. Three significant increases (p≤0.05 or p≤0.01) that did occur in the 140 or 430 mg/kg bw/day dosage groups were not considered related to Phenethyl Alcohol because they were slight increases and not reductions as might be expected. (Summaries - Tables B14 through B16).
Summary of Pup Alterations
In Groups I through IV, litters with pups with alterations numbered 16 (80.0%), 16 (80.0%), 16 (84.2%) and 2 (100.0%), respectively. The numbers of pups with any alteration observed were 42 (14.9%), 52 (18.8%), 50 (20.1%)* and 15 (62.5%)**, and the percentages of pups with any alteration per litter were 14.3%, 19.2%, 19.8% and 67.8%** in these same respective dosage groups. The significant increase in the percentage of pups with alterations in the 430 mg/kg bw/day dosage group was not considered related to maternal administration of Phenethyl Alcohol because the more relevant percentages of litters affected and the percentage of fetuses per litter with alterations were not significantly increased. Several of the 24 live pups in the two litters that had live pups in the 1400 mg/kg bw/day dosage group on postpartum day 21 had a skeletal alteration. These alterations included: short, small or incompletely ossified hyoid ala; a 7th cervical rib; bifid lumbar centrum; misaligned, fused, small or incompletely ossified caudal vertebrae; broad, proximate or short ribs; irregularly shaped manubrium; large sternal centra; irregularly shaped xiphoid or fused metacarpals. None of these fetuses will be discussed further. Individual observations for these fetuses are available in Table B29. All other pup alterations (malformations and variations) that occurred in the 0 (Vehicle Control), 140 and 430 mg/kg bw/day dosage group are summarized in the following text. (Summary - Table B14).
Pup Skeletal Alterations Malformations
No skeletal malformations occurred in any pup in the 0 (Vehicle Control), 140 or 430 mg/kg bw/day dosage group pups evaluated on day 21 postpartum
Variations-Hyoid
A total of 1, 1 and 7* pups from 1, 1, and 3 litters in the 0 (Vehicle Control), 140 and 430 mg/kg bw/day dosage groups, respectively, had a short hyoid ala. None of these pups had any other skeletal alterations. A total of 5, 1 and 7 pups from 4, 1, and 2 litters in the 0 (Vehicle Control), 140 and 430 mg/kg bw/day dosage groups, respectively, had a small hyoid ala. None of these pups had any other skeletal alterations.
Variations-Vertebrae
One pup in the 0 (Vehicle Control) dosage group had an arch in a cervical vertebra that had the appearance (shape and size) of the arch in the 7th cervical vertebra. No other skeletal alterations occurred in this pup. One pup in the 140 mg/kg bw/day dosage group had a bifid centrum in a thoracic vertebra. This pup had no other skeletal alterations. A total of 33, 45** and 32 pups from 13, 15 and 15 litters in the 0 (Vehicle Control), 140 and 430 mg/kg bw/day dosage groups, respectively, had one or more misaligned caudal vertebrae. No other skeletal alterations occurred in these pups. One pup in the 140 mg/kg bw/day dosage group had a fused caudal vertebra. No other skeletal alterations occurred in this pup. Four** pups from four** different litters in the 140 mg/kg bw/day dosage group had a small caudal vertebra. No other skeletal alterations occurred in these pups.
Variations-Ribs
One pup in the 140 mg/kg bw/day dosage group had a short rib. This pup had no other skeletal alterations. A total of 3, 3 and 6 pups each from a different litter in the 0 (Vehicle Control), 140 and 430 mg/kg bw/day dosage groups, respectively, had a thickened rib. No other skeletal alterations occurred in these pups. No other skeletal variations occurred.
Pup Ossification Site Averages
All apparent delays in ossification that were observed in the Caesarean-delivered fetuses in the 430 and 1400 mg/kg bw/day dosage groups were resolved by DL 21. The average number of ossified carpals in the forelimb was significantly reduced (p≤0.01) in the 1400 mg/kg bw/day dosage group, relative to the vehicle control group value (11.30 vs. 11.98 in vehicle controls). There were no other statistically significant or biologically important differences among the four dosage groups in the average numbers of ossification sites per pup for the hyoid, vertebrae (cervical, lumbar, sacral), sternum (manubrium, sternal centers and xiphoid), forelimbs (metacarpals and phalanges) or hindlimbs (tarsals, metatarsals, and phalanges). The statistically significant increase (p≤0.05 or p≤0.01) in the average number of ossified thoracic vertebrae, caudal vertebrae and rib pairs that occurred in the 430 mg/kg bw/day dosage group was considered unrelated to maternal treatment with Phenethyl Alcohol because the increases were not dosage-dependent. (Summaries - Tables B15 and B16) .
Necropsy Observations
No gross lesions related to treatment with Phenethyl Alcohol occurred. All necropsy observations were considered unrelated to Phenethyl Alcohol because: 1) the observations occurred in only one or two rats in any dosage group; and/or 2) the observation occurred in a rat that died prior to scheduled sacrifice. These gross lesions included a mottled appearance (red and dark red) to all lobes of the lungs, a rough appearance to the liver, tissue present in the stomach and a rough appearance to the kidneys which also had numerous large pitted areas that extended through the parenchyma out to the capsule. No other gross lesion occurred. (Summary - Table B17).
* Significantly different from the vehicle control group value (p≤0.05).
** Significantly different from the vehicle control group value (p≤0.01).
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 266 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The supporting study (read-across to PEA; CAS No. 60-12-8) is equivalent or similar to an OECD 414 study/GLP. The quality of the database is high.
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 140 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The key study (read-across to PEA; CAS No. 60-12-8) is equivalent or similar to an OECD 414 study/GLP. The quality of the database is high.
Additional information
There is no toxicity to reproduction (screening for reproductive/developmental toxicity) study available for DMBC.
There are five pre-natal developmental toxicity studies in rats available (read-across to PEA; CAS No. 60-12-8) - 2 oral studies via the diet (Politano et al., 2013) and gavage (Mankes et al., 1983) and 3 via the dermal route (Politano et al., 2013 and RIFM 58462). The RIFM 58462 study was chosen as the key study as (i) it was conducted according to a protocol that is equivalent or similar to OECD 414/GLP (ii) had an exposure period of DG 7 -20 with a second exposure group extended to natural delivery and sacrificed 21 days post-partum (iii) had sufficient number of pregnant rats for evaluation (apart from excessive toxicity noted at 1400 mg/kg bw/day) and (iv) had comprehensive documentation.
Key study
In a combined dermal developmental and perinatal/postnatal reproduction toxicity key study (RIFM 58462; equivalent or similar to OECD 414/GLP), PEA (99.42%) or deionized water was administered dermally (occlusive) to 40 female Crl:CD(SD) rats at dose levels of 0, 140, 430 and 1400 mg/kg bw/day beginning on day 7 of presumed gestation and continuing through day 20 of presumed gestation. Twenty rats per dosage group were Caesarean-sectioned on day 21 of presumed gestation and 20 rats per dosage group were allowed to deliver their litters and were sacrificed on day 21 postpartum. Skin irritation was assessed using the Draize method.
In the rats assigned to caesarean-sectioning, mortality was significantly increased at 1400 mg/kg bw/day, in comparison to the vehicle control group value. Five and three rats in the 1400 mg/kg bw/day dosage group were found dead or humanely euthanized, respectively. Most of these rats had decreased motor activity, impaired or lost righting reflex, ataxia, hunched posture, mild or moderate dehydration (based on skin turgor), red perivaginal substance and vocalization before death. In addition, observations consistent with primary irritancy (erythema and flaking) occurred in most of the female rats that died or were euthanized. These skin reactions and clinical signs are consistent with observations noted in rats that survived to scheduled sacrifice; therefore, the deaths were attributed to treatment with Phenethyl Alcohol. Observations consistent with primary irritancy occurred in female rats percutaneously administered Phenethyl Alcohol during the gestation period. These skin reactions included: flaking grade 2 (≥430 mg/kg bw/day) and erythema grades 1 through 3 and flaking grade 3 (1400 mg/kg bw/day). Each of these skin reactions, with the exception of erythema grade 3, occurred in significantly more rats in the 430 and/or 1400 mg/kg bw/day dosage groups, in comparison to the vehicle control group values. The onset and severity of the skin reactions was, in general, dependent on the dosage of Phenethyl Alcohol. A statistically significant loss in body weight, coupled with reduced or significantly reduced feed consumption values, occurred at 1400 mg/kg bw/day for the entire dosage period, as compared to the vehicle control group values. Absolute and relative feed consumption values at 1400 mg/kg/day were 78% and 94% of the vehicle control group value, respectively, for DGs 7 to 21. Overall, body weight gains and absolute feed consumption values were significantly reduced for the entire gestation period, as compared to the vehicle control group values. At 1400 mg/kg bw/day, the average maternal body weight was significantly reduced beginning on DG 8 and continuing until DG 21.
Pregnancy occurred in 16 to 20 rats in each dosage group. As a result of the increased mortality that occurred in the 1400 mg/kg bw/day dosage group, Caesarean-sectioning observations on DG 21 were based on 20, 18, 20 and 9 pregnant rats in the 0 (Vehicle Control), 140, 430 and 1400 mg/kg bw/day dosage groups, respectively. At 1400 mg/kg bw/day, postimplantation loss (i.e., early and late resorptions and the percentage of resorbed conceptuses per litter) was increased or significantly increased, in comparison to the vehicle control group values. Seven of the nine rats in this dosage group had total litter loss (i.e., 100% resorbed conceptuses). These increases in postimplantation loss resulted in an overall significant reduction in the averages for litter size and live fetuses at 1400 mg/kg bw/day, in comparison to the vehicle control group values (1.3 per litter vs. 14.6 per litter in vehicle controls). At ≥430 mg/kg bw/day, the average fetal body weight (total, male and female) was significantly lower than the concurrent vehicle control group values. These values were also below the range observed historically at the Testing Facility. The combined fetal weight at 430 and 1400 mg/kg bw/day was 91% and 48% of the vehicle control group value, respectively. As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, all 12 fetuses in the two litters that had live fetuses at 1400 mg/kg bw/day had one or more gross, soft tissue and/or skeletal alterations. These alterations included: gross alterations of the tail, limbs and body; soft tissue alteration of the eye, vessels and heart; and skeletal alterations of the skull, vertebral column, limbs, manubrium, sternal centra, clavicle, ribs, and pelvis. The limited number of fetuses and litters with live fetuses compared to the other dosage groups resulted in significantly increased fetal and litter incidences of each of these alterations. At 1400 mg/kg bw/day, the average number of ossification sites per fetus per litter was significantly reduced for caudal vertebrae, sternal centra, xiphoid, metacarpals, phalanges, metatarsals and phalanges. Corresponding to the reduction in fetal weight in the 430 mg/kg bw/day dosage group, ossification site averages for caudal vertebrae, fore- and hindlimb phalanges and metatarsals were also significantly reduced and below the historical control range for this Testing Facility. The number of litters and fetuses with a cervical rib present at the 7th cervical vertebrae was significantly increased at 430 mg/kg bw/day, as compared to the vehicle control group values. No gross lesions related to treatment with Phenethyl Alcohol occurred.
In rats assigned to natural delivery observations, one rat was found dead on DG 13, at 1400 mg/kg bw/day This death was presumed related to treatment with Phenethyl Alcohol because: 1) similar events occurred in the rats assigned to Caesarean-sectioned; and 2) the observation occurred in the high dosage group. All other rats assigned to the natural delivery phase of this study survived to scheduled sacrifice. Observations consistent with primary irritancy occurred during the gestation period and included: erythema grades 2 and 3, edema grade 2 and flaking grade 3 (1400 mg/kg bw/day); and flaking grades 1 and 2 (≥140 mg/kg bw/day). Each of these skin reactions occurred in an increased or significantly increased number of rats in the 140, 430 and/or 1400 mg/kg bw/day dosage groups, in comparison to the vehicle control group values. The onset and severity of the skin reactions was, in general, dependent on the dosage of Phenethyl Alcohol. Clinical signs attributed to treatment with Phenethyl Alcohol included: decreased motor activity, vocalization, mild or moderate dehydration (based on skin turgor), ataxia, ptosis, hunched posture, urine-stained abdominal fur, ungroomed coat, red perivaginal substance, grooming/chewing at the back, a red and/or raw appearance to the back: scab forming an open abrasion on the back, an abrasion on the back, lacrimation, scant feces, chromodacryorrhea and soft or liquid feces. The number of affected rats at 1400 mg/kg bw/day during the gestation period was significantly increased, in comparison to the vehicle control group values. Body weight gains were significantly reduced for the entire gestation dosage period and the entire gestation period in the 1400 mg/kg bw/day dosage group, as compared to the vehicle control group values. Body weight gains in the 1400 mg/kg bw/day dosage group were 38% of the vehicle control group value on DGs 7 to 21. The average maternal body weight at 1400 mg/kg bw/day was significantly reduced beginning on DG 8 and continuing until DG 21. At 1400 mg/kg bw/day, the average maternal body weight was 82% of the vehicle control group value on DG 21. In addition, body weight gains were significantly reduced in the 430 and 1400 mg/kg bw/day dosage groups on Days 1 to 4 of lactation. Thereafter, body weight gains were comparable among the dosage groups. Absolute and relative feed consumption values were reduced or significantly reduced at 1400 mg/kg bw/day on DGs 7 to 21, in comparison to the vehicle control group values. Absolute and relative feed consumption values in the 1400 mg/kg bw/day dosage group were 82% and 94% of the vehicle control group value, respectively, for the entire dosage period. Corresponding to reductions in body weight gain, absolute and relative feed consumption values were also reduced at 1400 mg/kg bw/day dosage group on DLs 1 to 4 and overall for DLs 1 to 14, in comparison to the vehicle control group values.
Pregnancy occurred in 16 to 20 female rats in the four dosage groups. All pregnant dams at 140 and 430 mg/kg bw/day delivered litters, 8 of the 16 pregnant rats delivered a litter at 1400 mg/kg bw/day. Of these 8 dams, one had a litter with no liveborn pups and four had all pups die before day 4 postpartum. At 1400 mg/kg bw/day, the duration of gestation was significantly increased (24.0 days vs. 23.0 days in vehicle controls). In addition, the gestation index at 1400 mg/kg bw/day was significantly reduced, as compared to the vehicle control group value (40% vs. 100% in vehicle controls). The averages for the total number of pups delivered and the number of liveborn pups were reduced or significantly reduced in this same dosage group, in comparison to the vehicle control group values. The percentage of pups that died or were presumed cannibalized on day 1 and days 2 to 4 postpartum was significantly increased at 1400 mg/kg bw/day, as compared to the vehicle control group values. Reflecting this increase in pup mortality, the viability index at 1400 mg/kg bw/day was significantly reduced relative to the vehicle control group value (63.2% vs. 99.0% in vehicle controls). The average number of surviving pups per litter was significantly reduced at 1400 mg/kg bw/day on days 4, 7, 14 and 21 postpartum, in comparison to the vehicle control group values. In addition, the average pup weight per litter was significantly lower at 1400 mg/kg bw/day on day 1 postpartum relative to the vehicle control group value. On postpartum days 4, 7 and 21, pup body weights at 1400 mg/kg bw/day were 9%, 10% and 7% lower than the corresponding vehicle control group values. At 1400 mg/kg bw/day, a significant number of litters had pups with mild dehydration (based on skin turgor), a thread-like tail and were not nursing. As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, several of the 24 live pups in the two litters that had live pups at 1400 mg/kg bw/day on postpartum day 21 had a skeletal alteration. The small number of pups and litters resulted in many of these findings occurring at a significantly increased incidence, as compared to the vehicle control group values. These alterations included: short, small or incompletely ossified hyoid ala; a 7th cervical rib; bifid lumbar centrum; misaligned, fused, small or incompletely ossified caudal vertebrae; broad, proximate or short ribs; irregularly shaped manubrium; large sternal centra; irregularly shaped xiphoid or fused metacarpals. The average number of ossification sites per pup per litter was significantly reduced at 1400 mg/kg bw/day for carpals, in comparison to the vehicle control group value. All apparent delays in ossification that were observed in the Caesarean-delivered foetuses at 1400 mg/kg bw/day were resolved by DL21.
The maternal NOAEL was 430 mg/kg/day. The 1400 mg/kg/day dosage caused mortality and reductions in body weight and feed consumption. The developmental NOAEL for pups Caesarean-delivered on day 21 of gestation is 140 mg/kg bw/day. Maternal dosages of 430 and 1400 mg/kg bw/day caused reductions in fetal weight with corresponding delays in fetal skeletal ossification and an increase in the incidence of cervical ribs. All 12 fetuses in the two litters that had live fetuses in the 1400 mg/kg bw/day dosage group had one or more gross, soft tissue and/or skeletal alterations. The 1400 mg/kg bw/day dosage also produced embryo/fetal lethality.
The NOAEL for viability and growth in the offspring naturally delivered and euthanized on day 21 of lactation is 430 mg/kg bw/day. The maternal dosage of 1400 mg/kg bw/day increased the incidence of perinatal mortality. As expected in dams that exhibited extensive maternal and embryo/fetal toxicity, several of the 24 live pups in the two litters that had live pups in the 1400 mg/kg bw/day dosage group on postpartum day 21 had a skeletal alteration. However, all apparent delays in ossification and increased incidences of a cervical rib at the 7th cervical vertebrae that were observed in the Caesarean delivered fetuses in the 430 mg/kg bw/day dosage group were resolved by DL 21.
Supporting studies
A series of pre-natal developmental toxicity supporting studies (oral: diet, two dermal studies) were described in Politano et al., 2013.
In a pre-natal developmental toxicity study (equivalent or similar to OECD 414/GLP), PEA was administered to 28 female Crl:COBSCD(SD)B rats/group in the diet at dose levels of 0, 1000, 3000, or 10 000 ppm (equivalent to 0, 83, 266 and 799 mg/kg bw/day) from days 6 through 15 of gestation. There were no clinical signs of toxicity or mortality at any dosage level. Feed consumption and body weight gains were reduced predominantly on the first 2 days of treatment at the highest dosage level (results were not statistically different). No other maternal effects were observed during the study. On GD 20, necropsy revealed that 27 of 28 control rats and 26 of 28 rats in each of the low, mid, and -dosage groups were pregnant with live fetuses. Litter parameters, as assessed by mean values for embryo-fetal loss, litter sizes, sex ratios, and litter and mean fetal weights, were equivalent in control and PEA-exposed groups. The incidence, type, and distribution of fetal malformations, anomalies, and skeletal variants were statistically unaffected by maternal exposure to PEA at concentrations as high as 10 000 ppm (799 mg/kg bw/day). However, at the highest dosage level, a marginal delay in fetal ossification might have occurred, as a slightly higher incidence of fetuses with incomplete ossification of some skeletal elements, particularly the sacrocaudal vertebral arches, was observed.
The maternal and developmental NOAELs were 266 mg/kg bw/day, based on a transient decrease in maternal feed consumption and body weight gains on GDs 6 and 7 and a slightly higher incidence of delayed ossification in some fetal skeletal elements.
In a pre-natal developmental toxicity study (equivalent or similar to OECD 414/GLP), PEA was administered dermally (occlusive) to 25-35 female Crl:COBSCD(SD)B rats/group at dose levels of 0, 140, 430 and 1400 mg/kg bw/day from days 6 through 15 of gestation. A total of 3 satellite animals in the low- and high-dosage groups were administered radiolabeled PEA on GD 15, after which urine was collected for 24 hours to determine absorption. Local irritation at the application site was observed in some rats early in the treatment period, especially in the high-dosage group. Signs of toxicity included: irritability, hunched posture, walking on toes, pilo erection, and periorbital staining (these effects were restricted almost entirely to dams in the high-dosage group) but resolved by termination. Mean feed consumption and body weight gains were markedly reduced in the high-dosage group during the treatment period. Reversal of these effects occurred upon cessation of treatment, but mean body weights were still statistically depressed at termination. On GD 20, necropsy revealed that 21 of 25 control rats and 30, 22 and 18 rats in each of the low, mid, and -dosage groups were pregnant with live fetuses. There were no significant differences between control and test groups in mean liver or kidney weights, pregnancy rates, mean preimplantation losses, or gross changes at necropsy on GD 20. Clinical pathology determinations conducted in control and high-dosage dams revealed no adverse hematological or serum chemistry findings. In the absorption study, 63% to 66% of the dosage had been absorbed. Absorption was rapid and extensive, but not dose dependent. In the high-dose group, embryo-fetal toxicity was manifested as statistically significant increases in post-implantation loss with a statistically significant reduction in mean litter size and a marked depression of mean fetal body weight (2.08 g versus 3.26 g in control group, 3.33 g in the 140 mg/kg bw/day group, and 3.18 g in the 430 mg/kg bw/day group). None of these changes occurred at the 2 lower dosages. When fetuses were examined for soft tissue and skeletal abnormalities, a dose-related increase in defects was observed. Statistical analysis of incidences of abnormalities was considered unnecessary given the results obtained. At the highest dosage (1400 mg/kg), morphological changes were present in 160 of the 161 fetuses; the variety, incidence, and degree of the skeletal and soft tissue changes varied considerably among individual fetuses. However, high incidences of the following specific types of defects were noted in more than 40% of the fetuses and 70% of the litters at the high dosage (1400 mg/kg bw/day): anophthalmia or microphthalmia, ventricular septal defects, defects or irregularities affecting thoracic, lumbar, and sacrocaudal vertebrae (associated with short or kinky tail), defects of the thoracic ribs, and occurrence of rudimentary cervical ribs (RCR). At the mid-dosage (430 mg/kg bw/day), occasional fetuses displayed the specific changes observed at 1400 mg/kg bw/day, and the number of fetuses with RCR, thoracic changes, or moderate degrees of reduced ossification exceeded concurrent controls. At the low dosage (140 mg/kg bw/day), findings were equivocal; incidences of RCR and thoracic vertebral irregularities were only marginally higher than those in the controls.
In a corroborative pre-natal developmental toxicity follow up study (equivalent or similar to OECD 414/GLP), PEA was administered dermally (occlusive) to 10 female Crl: COBSCD (SD)BR Charles River rats/group at dose levels of 0, 70, 140, 280, 430 and 700 mg/kg bw/day from days 6 through 15 of gestation. No deaths occurred in the study, but all dosages produced localized, low levels of erythema and/or desquamation at the intrascapular application sites. The severity of these reactions was dose related. Additional signs of toxicity included ptosis and/or urine stained abdominal fur, noted only in dams at the 700 mg/kg bw/day dosage level. Classic signs of maternal toxicity, that is, reduced feed intake and body weight gains, did not occur in a consistent manner, which is often the case when the primary effects are associated with local irritation. Dosages as high as 700 mg/kg bw/day did not adversely affect average litter sizes, numbers of implantations, live fetuses, or post-implantation loss (resorptions). Fetal effects included dose-dependent reductions of the litter mean for live fetal body weights. The reductions were statistically significant at ≥ 140 mg/kg bw/day but not at the lowest dosage of 70 mg/kg bw/day. A significantly increased incidence of RCR occurred at 700 mg/kg bw/day; and minimal increases of reversible delays in ossification were observed at ≥70 mg/kg bw/day. The incidences of the reversible delays in ossification were within the historical ranges of the Testing Facility. Specific delays included: delayed sternal and/or pelvic ossification (all dosage levels); incompletely ossified (hypoplastic) ribs and/or wavy ribs at 70 and at ≥430 mg/kg bw/day; and delays in ossification of the metacarpals and hindpaw phalanges (≥140 mg/kg bw/day), caudal vertebrae (≥280 mg/kg bw/day), forepaw phalanges (≥430 mg/kg bw/day), and sternal centers (700 mg/kg bw/day). The combined results of these two studies indicate maternal and developmental NOAELs of 70 mg/kg bw/day, based on minimal dermal irritation in the dams and small (not statistically significant) reductions in fetal body weights at this dosage level. The decreases in fetal body weights and consequent reversible delays in ossification at 140 mg/kg bw/day and higher were probably associated with maternal stress produced by the localized dermal irritation at the application site. Delays in ossification are considered fetal variations (reversible delays in development) in rodents that are usually related to reduced fetal body weight and, in the absence of fetal malformations, are not considered a significant hazard to humans.
Disregarded study
In a disregarded developmental toxicity study (Mankes et al., 1983; equivalent or similar to OECD 414 ) PEA was administered to 5-7 female Long Evans rats by gavage at dose levels of 0, 4.3, 43 and 432 mg/kg bw/day from days 6 through 15 of gestation.
Signs of severe maternal intoxication were noted immediately following ingestion at 432 mg/kg bw/day. These signs persisted overnight and were observed after each daily dose. Lower levels (43 or 4.3 mg/kg bw/day were asymptomatic. One dam (432 mg/kg bw/day) died from intratracheal intubation. All offspring (100%) at 432 mg/kg bw/day were either dead or malformed. Lower levels caused fewer adverse effects in the offspring (97% at 43 mg/kg bw/day, 55% at 4.3 mg/kg bw/day). Intrauterine growth retardation occurred at 432 mg/kg bw/day and at 4.3 mg/kg bw/day. Average pup weight and crown-rump length were decreased (pup weight from 3.6 g in controls to 2.5 g at 432 mg/kg bw/day and 3.1 g at 4.3 mg/kg bw/day; and length from 3.7 cm in controls to 3.2 cm at 432 mg/kg bw/day and 3.5 cm at 4.3 mg/kg bw/day) on a per litter basis. Increased numbers (32 at 432 mg/kg bw/day and 5 at 4.3 mg/kg bw/day) of grossly runted (weight less than 2.7 g) live offspring were also noted in these groups. Embryolethality did not occur at 432 mg/kg bw/day. Mean litter size was decreased to 9 pups in at 43 mg/kg bw/day and at 4.3 mg/kg bw/day, as compared to 12 pups in control litters. This difference was not a significant decrease at 43 mg/kg bw/day due to the large (standard deviation = 7) variability. The 43 mg/kg bw/day fetal death rate (total of resorptions and stillborn) was increased significantly to 18% (compared to 6% in controls). A clear dose-related increase in malformations was caused by 2-phenylethanol. At the lowest (4.3 mg/kg bw/day) exposure group, 30 of 60 live births in all 7 litters were malformed; at 43 mg/kg bw/day, 93% were malformed from 7 of 7 litters; and at the highest exposure group (432 mg/kg bw/day), all 51 live offspring from 4 of 4 litters were grossly malformed. At 432 mg/kg bw/day, pups had malformed eyes (31 of 51), malformed limbs (21 of 51), hydronephrosis (15 of 51), neural-tube defects (12 of 51) and malformed digits (6 of 51). Figure 1 shows a grossly malformed pup at 432 mg/kg bw/day; note the micromelia, club and web foot, microphthalmia, and open eyes. Offspring from 43 mg/kg bw/day had malformed eyes (28 of 65), hydronephrosis (19 of 65), and limb defects (3 of 65). Eye defects (9 of 60) and hydronephrosis (5 of 60) occurred in 4.3 mg/kg bw/day pups. Reduced cranial-bone ossification occurred in all groups. Limb ossification was markedly reduced in micromelic pups at 432 mg/kg bw/day and at 43 mg/kg bw/day. The radius, ulna, and humerus appear to be extremely short with no cartilage. Other bony defects included an overall reduction in size (micrognathia), and lack of pelvis, tail bones, carpals, or tarsals. Variant (reduced) ossification of the rib and tail were noted in 16 43 mg/kg bw/day pups. Sternebral variations were noted in 5 of 60 4.3 mg/kg bw/day pups.
This pre-natal development toxicity study in the rat is not acceptable and does not satisfy the guideline requirement for this oral study (OECD 414) in rats. The number of pregnant rats were 5-7 per group, it was not conducted according to GLP and many maternal parameters are not described in the publication.
Predicted dose descriptors:
The maternal and developmental NOAEL (oral: diet) predicted for DMBC is 266 mg/kg bw/day based on a transient decrease in maternal feed consumption and body weight gains on GDs 6 and 7 and a slightly higher incidence of delayed ossification in some fetal skeletal elements.
The maternal NOAEL (dermal) predicted for DMBC is 430 mg/kg/day. The developmental NOAEL (dermal) predicted for DMBC for pups Caesarean-delivered on day 21 of gestation is 140 mg/kg bw/day. The NOAEL for viability and growth (dermal) predicted for DMBC in the offspring naturally delivered and euthanized on day 21 of lactation is 430 mg/kg bw/day with the any developmental effects resolved by day 21 of lactation.
Justification for classification or non-classification
Based on the available information in the dossier, the substance DMBC (CAS No. 100-86-7) does not need to be classified for reproductive toxicity when the criteria outlined in Annex I of 1227/2008/EC are applied, based on the series of read-across studies from PEA (CAS No. 60-12-8).
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