2-phenylethanol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, guideline study not available. Study acceptable for assement.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The requirments of the U.S Food and Drungs Administration (FDA) have been used as the basis for this study design.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phenylethyl alcohol

IUPAC Name:

Phenylethyl alcohol

Specific details on test material used for the study:

Name: PEA ( Phenylethyl Alchol)
CAS: 60-12-8
Description: Clear and free liquid (clear colorless liquid)
Lor/Batcg Number: 8EFN05
Dates Received: 03/07/2008 and 30/07/2008
Expiration Date: 25 April 2010
Storage: Room Temmperature
Supplier: Millennium Specialty Chemicals, Jacksonville, FL
Special Handling Instructions: Gloves, dust-mist/HEPA-filtered mask, and protective clothing were worn during formulation preparation and dosage. The bulk test article was handled in a chemical fume hood.

Test animals

Species:

rat

Strain:

Crj: 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
- Weight at study initiation: 215-250
- Fasting period before study: No reported
- Housing: F1 generation animals were individually housed in stainless steel wire- bottomed cages except during the cohabitation and postpartum periods.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Rats were given ad limitum access to Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO) in individual feeders.
- Water (e.g. ad libitum): Local water was freerly available to rats ad libitum from an automatic watering access system and/or individual water bottle attached to the cages. Water was filtered through a reverse osmosis membrane (R.O water). Chlorine was added to the processed water as bacteriostat.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 °C to 26 °C.
- Humidity (%): 30% to 70%
- Air changes (per hr): Minimum 10 changes
- Photoperiod (hrs dark / hrs light): Automatically controlled 12-hours light dark fluorescent light cycle. Each dark period beganat 19.00 hours

Administration / exposure

Route of administration:

dermal

Vehicle:

other: water reverse osmosis membrane processed deionized water (R.O. deionized water)

Details on exposure:

Before application each day, the skin site was graded for reactions (edema, erythema, eschar formation) and each rat was fitted with an Elizabethan collar to prevent oral ingestion of the vehicle and test material. Separate glass rods were used to apply each dosage level. The skin application site was occluded with aluminium foil, and then appropriately secured with micropore tape to prevent oral ingestion and evaporation. After 24-hr exposure period, the administration site was rinsed and then blotted dry.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

From day 7 to day 20 of presumed gestation.

Frequency of treatment:

Once daily

Duration of test:

From day 7 to day 20 of presumed gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

143 mg/kg bw/day (nominal)

Remarks:

0.14 mL/Kg bw/d

Doses / concentrations 3

Dose / conc.:

439 mg/kg bw/day (nominal)

Remarks:

0.43 ml/kg bw/d

Doses / concentrations 4

Dose / conc.:

1 428 mg/kg bw/day (nominal)

Remarks:

1.40 ml/kg bw/d

No. of animals per sex per dose:

Total: 160
40 rats per sex, per group.

Control animals:

yes

Details on study design:

0.14, 0.43, 1.40 ml/kg bw/d (nominal conc. (equivalent to 143, 439, 1428 mg/kg bw/d (based on d = 1.0202 g/cm3)))

Examinations

Maternal examinations:

The following parameters were examinated:
viability, skin reactions, clinical observations, body weights and body weight gains, feed consumption, Caesarean-sectioning observations, natural delivery observations, necropsy observation, foetal gross external, soft tissue ans skeletal alterations, including ossification site averages and pup sheletal alterations, including ossification site averages.

Ovaries and uterine content:

- Uteri of apparently nonpregnant rats: examined to confirm the absence of implantation sites.
- Uteri and ovaries of apparently nonpregnant rats: discarded at the end of the study when authorized by the Study Director.
- The number and distribution of corpora lutea: recorded.
- The uterus of each rat: 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.

Fetal examinations:

- Weigh (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam).
- Viability: at least twice daily.
- The pups in each litter were counted once daily.
- Clinical observations: once daily.
- Pup body weights: DLs 1, 4, 7, 14 and 21 (prior to sacrifice).
- Sex and gross external alterations.
- Visceral and skeletal alterations

Statistics:

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). The Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance were used 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 Test was 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 were greater 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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Rats Assigned to Caesarean-Sectioning

Mortality: At 1400 mg/kg/day, mortality was significantly increased, in comparison to the vehicle control group value. After 3 to 11 dosages, 5 and 3 rats in the 1400 mg/kg/day dosage group were found dead or humanely euthanized, respectively. Decreased motor activity, impaired or lost righting reflex, ataxia, hunched posture, mild or moderate dehydration (based on skin turgor), red perivaginal substance and vocalization were observed before death.

Skin Irritation: Primary irritancy occurred in most of the female rats that died or were euthanized. These skin reactions included: flaking grade 2 (_430 mg/kg/day) and erythema grades 1 through 3 and flaking grade 3 (1400 mg/kg/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/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/day was significantly increased in comparison to the vehicle control group values. In addition, low incidences of respiratory distress, 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 (severity was not noted) and mucoid feces occurred at 1400 mg/kg/day during the dosage period.

Body Weight: at 1400 mg/kg/day, a statistically significant loss in body weight and reduced feed consumption values were observed

Litter loss: At 1400 mg/kg/day, postimplantation loss (i.e., early and late resorptions and the percentage of resorbed conceptuses per litter) was increased or significantly increased. At 430 mg/kg/day, the average fetal body weight (total, male and female) was significantly lower than the concurrent vehicle control group values.


Rats Assigned to Natural Delivery Observations

Mortality: At 1400 mg/kg/day, one rat was found dead on DG 13. This death was presumed related to treatment with Phenethyl Alcohol.

Skin irritation: Primary irritancy occurred during the gestation period and included: erythema grades 2 and 3, edema grade 2 and flaking grade 3 (1400 mg/kg/day);
and flaking grades 1 and 2 (_140 mg/kg/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/day dosage groups, in comparison to the vehicle control group values.

Clinical signs: 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/day during the gestation period was significantly increased, in comparison to
the vehicle control group values. Moreover, 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/day during the dosage period, and were also attributed to treatment with Phenethyl Alcohol.

Body weight: Significant reduction was observed in the 1400 mg/kg/day dosage group, as compared to the vehicle control group values. In addition, body weight gains were significantly reduced in the 430 and 1400 mg/kg/day dosage groups on Days 1 to 4 of lactation. Thereafter, body weight gains were comparable among the dosage groups.

Pregnancy: in 16 to 20 female rats in the four dosage groups. All pregnant dams at 140 and 430 mg/kg/day delivered litters, 8 of the 16 pregnant rats delivered a litter at 1400 mg/kg/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/day, the duration of gestation was significantly increased (24.0 days vs. 23.0 days 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/day, as compared to the vehicle control group values.
At 1400 mg/kg/day, the average number of surviving pups per litter was significantly reduced at 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/day on day 1 postpartum relative to the vehicle control group value.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

439 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Rats Assigned to Caesarean-Sectioning

At 1400 mg/kg/day, foetuses from dams that presented maternal and embryo/fetal toxicity had live fetuses 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 average number of ossification sites per fetus per litter was also significantly reduced for caudal vertebrae, sternal centra, xiphoid, metacarpals, phalanges, metatarsals and phalanges. In the 430 mg/kg/day dosage group, ossification site averages for caudal vertebrae, fore- and hindlimb phalanges and metatarsals were also significantly reduced. The number of litters and fetuses with a cervical rib present at the 7th cervical vertebrae was significantly increased at 430 mg/kg/day, as compared to the vehicle control group values.
No gross lesions related to treatment with Phenethyl Alcohol occurred.


Rats Assigned to Natural Delivery Observations

Clinical signs: At 1400 mg/kg/day, a significant number of litters had pups with mild dehydration (based on skin turgor), a thread-like tail and were not nursing.
Alterations: Several live pups at 1400 mg/kg/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/day for carpals, in comparison to the vehicle control group value. All apparent delays observed in the Caesarean- delivered foetuses exposed to 1400 mg/kg/day were resolved at D21.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

439 mg/kg bw/day (nominal)

Basis for effect level:

other: Based on delayed skeletal development on day 20 of pregnancy, fully resolved

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The maternal no-observable-adverse-effect- level (NOAEL) of Phenyl Alcohol is 430 mg/kg/day. The developmental NOEL is 140 mg/kg/day.

Executive summary:

The aim of the study was to determine the reversibility of skeletal alterations and delays in skeletal ossification following treatment of Crl:CD(SD) female rats with Phenyl ethyl alcohol from day 7 to day 20 of presumed pregnancy. The results showed that at 1400 mg/kg/day dosage, Phenyl ethyl alcohol caused mortality and reduction in maternal body weight and feed consumption. Therefore, the maternal no-observable-adverse-effect- level (NOAEL) of Phenyl Alcohol is 430 mg/kg/day. No effects were observed on the foetuses of the pups of dams treated with 140 mg/kg/day of Phenyl ethyl alcohol, therefore, the developmental NOEL is 140 mg/kg/day. Maternal dosages of 430 and 1400 mg/kg/day caused reductions in fetal weight in the Caesarean-delivered foetuses with corresponding delays in foetal skeletal ossification. A reduction of the pup body weights were also recorded at 1400 mg/kg/day dosage throughout the post-partum period. At dose level of 1400 mg/kg/day, embryo/fetal lethality and increased incidence of perinatal mortality were observed. In addition, one or more gross, soft tissue alteration (included an increase in the incidence of cervical ribs) in the Caesarean-delivered foetuses were recorded as well as skeletal alterations in pups on postpartum day 21. However, all apparent delays in ossification and increased incidences of a cervical rib at the 7^thcervical vertebrae that were observed in the Caesarean delivered fetuses in the 430 mg/kg/day dosage group were resolved by DL21.