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EC number: 200-456-2 | CAS number: 60-12-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Additional information
Short description of key information:
No information is available
Effects on developmental toxicity
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1983
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: No GLP-compliant, guideline study not available. Study acceptable for assement.
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- No Guideline available
- GLP compliance:
- not specified
- Species:
- rat
- Strain:
- Long-Evans
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: - Source: Blue Spruce Farm in Altamont, NY
- Housing: after mating the females were housed in wire-topped plastic cages and additional bedig material.
- Diet and water: ad libitum
- Acclimation period: minimum 2 weeks - Route of administration:
- oral: gavage
- Vehicle:
- other:
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: Aqueous suspensions of 2-phenylethanol were freshly made. Doses of 20 ml/kg per day were administrated to female rats based on their current weights.
EXPOSURE LEVELS: The exposure levels were chosen as fractional doses (24, 2.4, or 0.24%) of the available LD50 values for Phenylethyl Alcohol, 1 log interval apart. - Details on mating procedure:
- - Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- Duration of treatment / exposure:
- From 6 to 15 days of gestation
- Frequency of treatment:
- Once daily
- No. of animals per sex per dose:
- Group 1: 19 pregnant rats serving as controls
Group 2: 5 pregnant rats dosed with 432 mg/kg
Group 3: 7 pregnant rats dosed with 43 mg/kg
Group 4: 7 pregnant rats dosed with 4.3 mg/kg - Control animals:
- yes
- Details on study design:
- The exposure levels were selected as fractional doses (24, 2.4 or 0.24%) of the available LD50 values for 2-phenylethyl alcohol, 1 log interval apart (Owston et al., 1981).
- Maternal examinations:
- Dams were observed daily for signs of toxicity and lethality. Body weights were recorded on day 0-15 and 20 of gestation.
- Ovaries and uterine content:
- -Total uterine weights and total litter weights were observed and recorded at day 20.
- Fetal examinations:
- At day 20 the following observations were recorded: individual pup weights, number of live pups, stillbirths, resorptions, implantations,sites, sex distributions, crown-rump length and number of corpora lutea.
All pups were examined for gross malformations at birth: soft-tissue (internal) defects and skeletal variations were detected using the Wilson method (1965) and the McLeod method (1980), respectively. - Statistics:
- All data generated were statistically analysed on a DEC system 10 computer. The method Dixon and Brown (1977) was used to perform statistical analysis. Litters and fetus were both used as experimental units. 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, total dead and malformed, and incidences of malformations (Mankes et al., 1981).
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Signs of severe maternal intoxication were noted immediately following administration of 432 mg/kg/bw. The signs were observated after ach daily dose. Dosage levels of 43 and 4.3 mg/kg/bw were asymptomatic. - Dose descriptor:
- NOAEL
- Effect level:
- 43 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- LOAEL
- Effect level:
- 4.3 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
REPRODUCTIVE EFFECTS
Group 2: 432 mg/kg/bw
100% of the pups were dead or malformated- Intrauterine growth retardation- Decreased average pup weight and crown-rump length (weight: 3.6 g in controls to 2.5 g of pups under treatment), length (3.7 cm of controls and 3.2 cm of pups under treatment)
- Increased numbers of grossy runted (weight less than 2.7 g) live offspring (32)
Group 4: 4.3 mg/kg/bw
- Intrauterine growth retardation
- Decreased average pup weight and crown-rump length (weight: 3.6 g in controls to 3.1 g of pups under treatment), length (3.7 cm of controls and 3.5 cm of pups under treatment)
- Increased numbers of grossy runted (weight less than 2.7 g) live offspring (5)
EMBRYOLETHALITY
- No embryolethality at 432 mg/kg/bw
Group 3: 43 mg/kg/bw
- Decreased mean litter size to 9 pups. No stastically difference due to the large standard deviation.
- Fethal death rate was increased significantly to 18% (compared to 6% in controls)
Group 4: 4.3 mg/kg/bw
- Decreased mean litter size to 9 pups.
TERATOLOGY
Group 2: 432 mg/kg/bw
- all 51 live offspring from 4 of 4 litters were grossly malformated
- eye, limbs, hydronephrosis, neural-tube defects and digits malformations
- reduced cranial-bone ossification
- limb ossification was markedly reduced in micromelic pups
Group 3: 43 mg/kg/bw
- 93% were malformated
- eye, limbs, hydronephrosis malformations
- limb ossification was markedly reduced in micromelic pups
- variant (reduced) ossification of the rib and tail were noted in 16 pups
Group 4: 4.3 mg/kg/bw
- 30 of 60 live births in all 7 litters were malformated
- eye and hydronephrosis malformations
- reduced cranial-bone ossification
- sternebral variations were noted in 5 of 60 pups - Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- The highest no observed adverse effect level (NOAEL) for maternal toxicity is 43 mg/kg/bw/day. The NOEAL for development, teratogenecy and fetotoxicity is 4.3 mg/kg/bw/day
- Executive summary:
The author investigated the effects of Phenyl ethyl Alcohol (PEA) in pregnant Long-Evans rats. The animals were treated with 4.3, 43, 430 mg/kg/day by gavage of PEA dissolved in aqueous solution, from day 6 to day 15 of gestation. At day 20 fetuses were delivered by Caesarean section. Dams were observed daily for sign of toxicity and lethality and all pups were examined for gross malformation. Fetotoxicity and embryolethality were also evaluated. The results showed that the highest dosage level, 432 mg/kg/bw/day caused maternal toxicity. However, 43 and 4.3 mg/kg/bw/day resulted symptomatic. Observations in the offspring showed that all dosage-levels caused malformations such as intrauterine growth retardation
, decreased average pup weight and crown-rump length, and increased numbers of grossly runted. No embryolethality was observed at 432 mg/kg/bw. However, fetal death rate was increased significantly to 18% (compared to 6% in controls) at 43 mg/kg/bw/day. Most of the signs observed are dose-level dependent. In conclusion, 2 -phenylethyl alcohol induces maternal toxicity at dosage level of 432 mg/kg/bw/day and teratogenicity, embryolethality and developmental toxicity already at 4.3 mg/kg/bw/day.
- 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.
- 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
- 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. - 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 - 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
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 143 mg/kg bw/day (nominal)
- Remarks:
- 0.14 mL/Kg bw/d
- Dose / conc.:
- 439 mg/kg bw/day (nominal)
- Remarks:
- 0.43 ml/kg bw/d
- 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)))
- 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. - 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. - Dose descriptor:
- NOAEL
- Effect level:
- 439 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- 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. - 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
- Developmental effects observed:
- not specified
- 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 7thcervical vertebrae that were observed in the Caesarean delivered fetuses in the 430 mg/kg/day dosage group were resolved by DL21.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1988
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP-compliant, guideline study not available. Study acceptable for assement.
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- No Guideline available
- GLP compliance:
- yes
- Species:
- rat
- Strain:
- Crj: CD(SD)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York
- Age at study initiation: 61 days
- Weight at study initiation: 157- 205 grams
- Fasting period before study:
- Housing: The female rats were housed individually in wire- bottomed stainless steel cages (11x9x8 inches) suspended above absorbet paper liners.
- Diet (e.g. ad libitum): Certified Rodent Chow 5002M (Ralston Purina) available ad limitum.
- Water (e.g. ad libitum):Local water filtered through osmosis membrane was available ad libitum.
- Acclimation period: 3 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70
- Humidity (%): 48-58
- Air changes (per hr): 10 changes per hour of 100% fresh air filtered with HEPA- filteres (Airo Clean room) at 99.97%
- Photoperiod (hrs dark / hrs light): automatically controlled fluorescent light cyclo was maintained at 12 hours light and 12 hours dark, with each dark cycle beginning at 19.00 hours EST.
IN-LIFE DATES: From: To: - Route of administration:
- dermal
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- Prior carrying out the test, the exposure area was shaved using electric clippers. The shaved area was approximately 7 cm x 5 cm on the intrascapular region of each animal. The exposure areas were clipped as necessary during the dosage period. Prior the test, the shaved area was mapped to exclude skin alterations. To minimise evaporation of the test material, the treated area was occluded by a dressing (5 cmx 5cm) consisting of an aluminium foil patch that was held in place by a medical- type adhesive bandage.
The dressing was placed on the animals immediately after application of the test material and was not removed for approximately 24 hours.
Control rats were treated with a ml/kg volume of reverse osmosid membrane processed deionized water that was equivalent to the 0.70 ml/kg dosage. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Dosage level were adjusted based on body weights.
- Details on mating procedure:
- After the acclimation period, 130 of the 148 apparently healthy virgin female rats were placed in cohabitation with male breeder rats for a maximum of 4 days (1 male was paired with & female rat). Females with spermatozoa observed in vaginal smears, or a copulatory plug observed in the vagina or in the cage pan, were considered to be at day 0 of the presumed gestation and assigned to individual housing.
- Duration of treatment / exposure:
- Treatment on gestation days 6-15
- Frequency of treatment:
- Once daily
- Duration of test:
- Termination on gestation day 20
- Dose / conc.:
- 0.07 other: ml/kg/day
- Dose / conc.:
- 0.14 other: ml/kg/day
- Dose / conc.:
- 0.28 other: ml/kg/day
- Dose / conc.:
- 0.43 other: ml/kg/day
- Dose / conc.:
- 0.7 other: ml/kg/day
- No. of animals per sex per dose:
- 8 female
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The dosage-range study was designed to determine:
1. wheater cervical ribs, particularly those definid as "vestigial", were the most sensitive indicator of developmental toxicity;
2. wheater this alteration occurred in the absence of both maternal and or other developmental effects;
3. definitive maternal and developmental toxicity no-effect levels. - Maternal examinations:
- VIABILITY
The rats were observed 2 times per day
CLINICAL SIGNS and GENERAL APPEREANCE
Observations during acclimation, cohabitation and pre-dosage (day 1 to day 5 of presumed gestation) periods, from day 6 to day 15 of presumed pregnancy and approximately 30 minutes after administration. All females that mated were evaluated on day 0 of presumed gestation.
Rats were observed also for abortion and natural deliver.
DERMAL IRRITATION and SCORING
Daily incidence of skin irritation using erythema and edema endpoint by Draize and the National Research Council were recorded.
BODY WEIGHT and FOOD CONSUMPTION
The body weights and food consumption of female rats were recorded 4 times prior the study, on day 0 and daily from day 0 to day 20 of gestation. - Ovaries and uterine content:
- NECROSPY
At day 20 each dam was sacrificed and examined for gross external and visceral lesions.
Corpora lutea in each ovary were identified and counted. The uterus of each rat was examined for pregnancy, number and placement of implantation, early and late resorption. - Fetal examinations:
- FETAL EVALUATIONS
Sex and gross external alteration were recorded. Approximately one-third of live foetuses were used to evaluate visceral alteration, using the Staples technique. A single free- hand cross- section of the skull was made in order to evaluate the presence of cervical ribs.
All other foetuses were examined for skeletal alterations, ossification per litter as well as late resorptions and dead foetuses. - Statistics:
- A parametric and non-parametric stastistical test was done. For all evaluation, minimum level of significance reported was p<=0.05.The variance test was used to analyse maternal physical sign data and the incidence of pregnancy, abortion, death and total resorption.
If the Analysis of variance was significant and appropriate (it passes the Bartlettù s test) then Dunnettùs test was used to identify the statistical significance of each gropus. If the Analysis of Variance was not appropriate, the Kruskal- Wallis test was used. - Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
MORTALITY
No dead rats occurred in the study.
ADVERSE CLINICAL SIGNS
After application of 0.70 ml/kg/day ptosis for 6 rats of the highest dosage level was observed. Urine- stained abdominal fur was also recorded for 3 rats.
SKIN REACTION
Grade 1 of erythema and/or desquamation was observed in all rats of all groups. The severity of the effect was dosage-dependent.
NECROSPY
Skin lesions were observed in 3 rats treated with 0.70 ml/kg/day and 1 treated with 0.28 ml/kg/day. The effects depended on the dermal application of neat PEA.
No other lesions were observed after administration of PEA.
MATERNAL BODY WEIGHT
A small inhibitory effect on average maternal body weight gain occurred for dams given the 0.14, 0.43 and 0.70 ml/kg/day.
MATERNAL FEED CONSUMPTION
Average maternal feed consumption for days 12-13 of gestation decreased significantly at 0.70 ml/kg/day
CAESAREAN- DELIVER DATA
No pregnant dams resorbed all of its conceptuses. - Dose descriptor:
- NOAEL
- Effect level:
- 714 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 71 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
MALFORMATION
Cervical ribs occurred only at dosage level of 0.70 ml/kg/day.
DELAYED OSSIFICATION
Incompletely ossified ribs occurred at concentartion level of 0.043 and 0.70 ml/kg/day.
Incompletely ossified sternal centers occurred at all concetration levels. No adverse effects were observed in the controls.
Delayed ossification of the pelvis were observed at 0.07, 0.28, 0.43, 0.70 ml/kg/day. No effects were observed in the controls and no dosage- dependent relationship was demostred.
FOETAL OSSIFICATION SITES
Ossified metacarpals and ossified hindpaw phalanges per foetus averaged occurred at dosage level of 0.14, 0.28, 0.43, 0.70 ml/kg/day.
Ossified caudal vertebrae per foetus occurred at 0.28, 0.43, 0.70 ml/kg/day, while foetuses with ossified forepaw phalanges occurred only at 0.43, 0.70 ml/kg/day.
Other foetal ossifications were observed, but werenot dosage-dependent and not significant different from the control groups. - Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- In a developmental toxicity study, 2 phenylethyl alcohol was administered dermally to female Crj: CD(SD) rats at dose levels of 0, 0.07, 0.14, 0.28, 0.43 and 0.70 ml/kg/day. The maternal and developmental NOAEL is 0.7 ml/kg/day. The foetal and developmental toxicity NOEAL is 0.07 ml/kg/day.
- Executive summary:
The test material Phenyl ethyl alcohol was administrated dermally to 8 female Crj: CD(SD) rats at dose levels of 0, 0.07, 0.14, 0.28, 0.43 and 0.70 ml/kg/day. The dosage-range study was designed to determine:1. wheater cervical ribs, particularly thosedefinedas "vestigial", were the most sensitive indicator of developmental toxicity, 2. wheater this alteration occurred in the absence of both maternal and or other developmental effects, 3. definitive maternal and developmental toxicity no-effect levels. The results of the study shows that no dead rats dead rats occurred in the study. After application of 0.70 ml/kg/day, ptosis for 6 rats of the highest dosage level was observed. Urine- stained abdominal fur was also recorded for 3 rats. In the application area, a grade 1 of erythema and/or desquamation was observed in all rats of all groups. The severity of the effect was dosage-dependent. Skin lesions were observed in 3 rats treated with 0.70 ml/kg/day and 1 treated with 0.28 ml/kg/day. The effects depended on the dermal application of neat PEA. Concerning the fetuses, cervical ribs occurred only at dosage level of 0.70 ml/kg/day. Incompletely ossified ribs occurred at concentration level of 0.43 and 0.70 ml/kg/day. Incompletely ossified sternal centers occurred at all concentrationlevels. No adverse effects were observed in the controls. Moreover, delayed ossification of the pelvis were observed at 0.07, 0.28, 0.43, 0.70 ml/kg/day. No effects were observed in the controls and no dosage- dependent relationship wasdemostred.
Ossified metacarpals and ossified hindpaw phalanges per foetus averaged occurred at dosage level of 0.14, 0.28, 0.43, 0.70 ml/kg/day.
Ossified caudal vertebrae per foetus occurred at 0.28, 0.43, 0.70 ml/kg/day, while foetuses with ossified forepaw phalanges occurred only at 0.43, 0.70 ml/kg/day. Other fetal ossifications were observed, butwere not dosage-dependent and not significant different from the control groups. In conclusion, the maternal NOAEL is 0.7 ml/kg/day, and the foetal and developmental toxicity NOEAL is 0.07 ml/kg/day.
- Endpoint:
- developmental toxicity
- Adequacy of study:
- supporting study
- Study period:
- 2017-08-21 to 2018-03-21
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Dose Range Finding Prenatal Developmental Toxicity Study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- yes
- Remarks:
- None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
- Principles of method if other than guideline:
- The design of this study is based on the following study guidelines, however the number of animals per group was only six instead of twenty-two:
• OECD 414, Prenatal Developmental Toxicity Study, 2001.
• (EC) No 440/2008, B.31 Prenatal Developmental Toxicity Study, 2008.
• EPA OPPTS 870.3700, Prenatal Developmental Toxicity Study, 1998. - GLP compliance:
- yes
- Remarks:
- Analyses to support the information cited in the Certificate of Analysis for the test item were not conducted in compliance with GLP regulations. These analyses were, however, conducted in compliance with Good Manufacturing Practice (GMP) regulations.
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Symrise; Batch No. 80100016
- Expiration date of the lot/batch: 2019-03-22
- Purity test date: 2017-06-19
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under test conditions: Stable until 22 March 2019 (expiry date)
- Solubility and stability of the test substance in the solvent/vehicle: Stability in vehicle (0.5% CMC + 1.25% Tween 80): Stability for at least 6 hours at room temperature, 8 days in the refrigerator and 3 weeks in the freezer is confirmed over the concentration range 0.5 to 200 mg/mL, Test Facility Study No. 519518.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
OTHER SPECIFICS:
Molecular formula: C8H10O
pH: 6.0-7.0
Specific gravity/density: 1.0180 (D25/25) - Species:
- rabbit
- Strain:
- New Zealand White
- Remarks:
- time-mated female New Zealand White rabbits
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 17-19 weeks old
- Weight at study initiation: 3153-3968 g (Groups 1-3) or 3162-4204 g (Groups 4-6)
- Fasting period before study: Not specified
- Housing: On arrival and following randomization females were housed individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles.
- Diet (e.g. ad libitum): Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during the study period.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles/containers.
- Acclimation period: at least 5 days before the commencement of dosing.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20°C (actual daily mean temperature during the study period)
- Humidity (%): 47-91% (actual daily mean relative humidity during the study period)
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
IN-LIFE DATES: From: Groups 1-3: 2017-08-21; Groups 4-6: 2018-01-26 To: 2018-02-23 - Route of administration:
- oral: gavage
- Vehicle:
- other: 0.5% CMC + 1.25% Tween 80 (Batch: 230817002, 150917001 and 290118001)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly as a suspension, formulated in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration in vehicle: Group 1 (Control): 0 mg/mL; Group 2: 2.15 mg/mL; Group 3: 21.5 mg/mL; Group 4 (Control): 0 mg/mL; Group 5: 100 mg/mL; Group 6: 150 mg/mL
- Amount of vehicle (if gavage): 2 mL/Kg
- Lot/batch no. (if required): 230817002, 150917001 and 290118001
Preparation of Vehicle
The vehicle, 0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80, was prepared at least monthly, stored in a refrigerator set to maintain 4°C. The prepared vehicle was removed from the refrigerator and stirred for at least 30 minutes before use. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analyses were performed using a validated analytical procedure (Test Facility Study No. 519518).
Concentration Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.
Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.
Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 519518) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. - Details on mating procedure:
- Time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France). The females arrived on Day 1 post-coitum (Day 0 post-coitum was defined as the day of mating).
- Duration of treatment / exposure:
- The test item and vehicle were administered once daily via oral gavage, 7 days a week from Day 6 to Day 28 post-coitum, inclusive.
- Frequency of treatment:
- once daily via oral gavage
- Duration of test:
- Day 6 to Day 28 post-coitum, inclusive
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- Group 1 (Control)
- Dose / conc.:
- 4.3 mg/kg bw/day
- Remarks:
- Group 2
- Dose / conc.:
- 43 mg/kg bw/day
- Remarks:
- Group 3
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- Group 4 (Control): included in the study based on the results of Groups 1-3
- Dose / conc.:
- 200 mg/kg bw/day
- Remarks:
- Group 5: included in the study based on the results of Groups 1-3
- Dose / conc.:
- 300 mg/kg bw/day
- Remarks:
- Group 6: included in the study based on the results of Groups 1-3
- No. of animals per sex per dose:
- 6 females/dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
For Groups 1-3, the dose levels were selected at request of the Sponsor and based on the results of a prenatal developmental toxicity study in rats (Mankes, R.F., LeFevre R., Bates H., Abraham R., Effects of various exposure levels of 2-phenylethanol on fetal development and survival in Long-Evans rats. Journal of Toxicology and Environmental Health, 12, 235-244 (1983)). Long-Evans rats were treated with 0, 4.3, 43 or 432 mg/Kg phenyl ethanol (CAS# 60-12-8) on Days 6-15 of gestation. At dose levels of 4.3 and 432 mg/Kg intrauterine growth retardation was noted. Embryolethality was 10% in the 4.3 mg/Kg dose group and 18% in the 43 mg/Kg dose group. The incidence of malformed fetuses was 100% at 432 mg/Kg, 93% at 43 mg/Kg and 50% at 4.3 mg/Kg; all litters in all dose groups were affected. Teratogenic effects of
phenyl ethanol in the offspring of Long-Evans rats manifested themselves in increased incidence of malformed eyes, neural tube defects, hydronephrosis and limb defects. Based on the above data dose levels of 0, 4.3 and 43 mg/Kg were selected initially for the dose range finder in pregnant rabbits, to determine whether this resulted in similar developmental toxicity as in the previous rat study.
As no clear developmental toxicity was observed in rabbits treated up to 43 mg/Kg, three additional groups (Groups 4-6) were added to the dose range finder in order to investigate the potential of the test material to induce developmental toxicity at higher dose levels. The dose levels of Groups 4-6 were selected based on the tolerability study in the non-pregnant rabbit (Test Facility Study No. 20138885, reported in Appendix 6 of the study report),which was conducted in between the experimental phases of Groups 1-3 and 4-6 of the dose range finder. As in this tolerability study, a maximum tolerated dose level in non-pregnant rabbits was established at 300 mg/Kg, dose levels of 0, 200 and 300 mg/Kg were selected for Groups 4-6 of the dose range finder in pregnant rabbits.
- Rationale for animal assignment (if not random): One day after receipt, animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the mean group.
- Justification for Test System and Number of Animals:
The New Zealand White rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for developmental toxicity testing by regulatory agencies. The laboratory (Charles River Den Bosch) has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test material, as clear developmental toxicity was expected based on the results of a previous performed prenatal developmental toxicity study in rats (Mankes, R.F., LeFevre R., Bates H., Abraham R., Effects of various exposure levels of 2-phenylethanol on fetal development and survival in Long-Evans rats. Journal of Toxicology and Environmental Health, 12, 235-244 (1983)).
This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives. Currently, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist. The study plan, including the addition of three extra groups, was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch (DEC 14-49 incl. amendment(s) and amendment 519517A) as required by the Dutch Act on Animal Experimentation (February 1997). - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy.
BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 2, 6, 9, 12, 15, 18, 21, 24, 27 and 29 postcoitum. In order to monitor the health status, animal no. 32 (Group 6) was also weighed on Day 11 post-coitum.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was quantitatively measured over Days 2-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29 post-coitum
Unscheduled Deaths:
If necessary for humane reasons, animals were euthanized as per Test Facility SOPs. These animals were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol® 20%), underwent necropsy, and specified tissues were retained.
Scheduled Euthanasia:
Animals surviving until scheduled euthanasia were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol® 20%) on Day 29 post-coitum. No terminal body weight was recorded.
Necropsy: All animals (including animals sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes (Each ovary and uterine horn of all animals was dissected and examined)
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: No
- Number of early resorptions: No
- Number of late resorptions: No
- Other: a) The number and distribution of live and dead fetuses; b) The number and distribution of embryo-fetal deaths - Fetal examinations:
- - External examinations: Yes: all per litter (Each viable fetus)
- Soft tissue examinations: Yes: all per litter
All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (Stuckhardt, J.L.; Poppe, S.M. Fresh visceral examination of rat and rabbit fetuses used in teratogenicity testing. Teratogenesis, Carcinogenesis and Mutagenesis,4, 181-188 (1984)). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (Woo, D.C., Hoar, R.M. Apparent hydronephrosis as a normal aspect of renal development in late gestation of rats: the effect of methyl salicylate. Teratology, 6, 191-196 (1972)).
- Skeletal examinations: Yes: all per litter
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson (Dawson, A.B. A note on the staining of skeletons of cleared specimens with Alizarin Red S. Stain Technology.1, 123-124 (1926). Subsequently, the skeletal examination was done on all fetuses from all groups.
- Head examinations: Yes: all per litter
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique (Wilson J.G. - Embryological Considerations in Teratology. In “Teratology: Principles and Techniques”; Wilson, J.G. and Warkany, J., Eds.; The University of Chicago Press: Chicago, IL, 251-277 (1965)). After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice. - Statistics:
- Please see 'Any other information on materials and methods incl. tables' for information on statistics.
- Indices:
- Maternal Variables
1) Body Weight Gains: Calculated against the body weight on Day 6 post-coitum
2) Corrected Body Weight Gains: Terminal body weight minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
3) Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Reproduction and Developmental Variables: For each group, the following calculations were performed:
1) Pre-implantation loss (%): ((number of corpora lutea - number of implantation sites) / (number of corpora lutea)) x 100
2) Post-implantation loss (%): ((number of implantation sites - number of live fetuses) / (number of implantation sites)) x 100
The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%): ((number of viable fetuses affected/litter) / (number of viable fetuses /litter)) x 100 - Historical control data:
- Historical Control Data of Fetal Examinations presented in Appendix 3 of the study report.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- In the 300 mg/Kg dose group, three females (nos. 32, 34 and 35) were sacrificed in extremis on Day 11 or 12 post-coitum before dosing on that day, as they had no food consumption from the start of treatment onwards resulting in a markedly body weight loss (14-17%), and absence of feces production. One of these females was additionally noted with lethargy, uncoordinated movements, a pale and lean appearance.
The remaining three females at 300 mg/Kg (nos. 31, 33 and 36) were noted with some slight signs of toxicity (body weight loss up to 2-5% and markedly reduced to no food consumption). Because of the severe toxic effects observed in the other half of the animals, these females were terminated early on Day 12 post-coitum due to animal welfare reasons.
Piloerection was noted for one female at 200 mg/Kg over Days 23-28 post-coitum onwards, which might be related to treatment with the test material. Abnormal licking was noted for all females of the 200 and 300 mg/Kg groups during the treatment period, which was considered to be related to the taste of the test materialformulations rather than a sign of systemic toxicity, taking into account the nature and minor severity of the finding at its time of occurrence (i.e. after dosing).
Reduced feces production (up to a severe degree) was noted for all females at 200 and 300 mg/Kg, compared to half of the females of the control, 4.3 and 43 mg/Kg groups. The high incidence at 200 and 300 mg/Kg was considered to be due to the significantly reduced food consumption. The incidence in the other groups was within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in the study.
Incidental findings of note were the fearful behavior in combination with vocalization observed for one female at 4.3 mg/Kg (no. 8) on Day 8 post-coitum, and exophthalmos and erythema of the right eye of one female of the 200 mg/Kg group (no. 27) during the pre-treatment and treatment period. These findings occurred in single females and as they were transient or were observed before initiation of treatment, they were not considered to be test material related.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain and were not considered to be toxicologically relevant. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Treatment at 300 mg/Kg (Group 6) resulted in preterm euthanasia of all six animals:
Three females (nos. 32, 34 and 35) were sacrificed in extremis on Day 11 or 12 post-coitum before dosing on that day, as they had no food consumption from the start of treatment onwards resulting in a markedly body weight loss (14-17%), and absence of feces production. One of these females was additionally noted with lethargy, uncoordinated movements, a pale and lean appearance.
The remaining three females at 300 mg/Kg (nos. 31, 33 and 36) were noted with some slight signs of toxicity (body weight loss up to 2-5% and markedly reduced to no food consumption). Because of the severe toxic effects observed in the other half of the animals, these females were terminated early on Day 12 post-coitum due to animal welfare reasons.
Macroscopic examination at necropsy for these animals revealed no findings that were considered to be test item-related. One female sacrificed in extremis (no. 34) was noted with isolated watery-clear cysts in the oviducts and one of the remaining females (no. 36) was noted with ectopic splenic tissue and scab formation on the lip.
No mortality occurred in any of the other groups. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Three females (nos. 32, 34 and 35) in the 300 mg/Kg group (sacrificed in extremis on Day 11 or 12 post-coitum before dosing on that day) did not consume food from the start of treatment resulting in a markedly body weight loss (14-17%) and absence of faeces production.
The remaining three females at 300 mg/Kg (nos. 31, 33 and 36; terminated early on Day 12 post-coitum due to animal welfare reasons) were noted with some slight signs of toxicity (body weight loss up to 2-5% and markedly reduced to no food consumption).
Treatment at 200 mg/Kg resulted in a significant body weight loss (average of 6%) over Days 6-9 post-coitum, followed by a statistically significantly reduced body weight gain, when compared to the concurrent control group. This resulted in statistically significantly lower mean body weights over Days 9 to 27 post-coitum (differences up to 10%), when compared to the concurrent control group. The mean corrected body weight gain for gravid uterus at 200 mg/Kg was slightly higher than the other groups. This was not regarded to be toxicologically relevant, as there was a high variation in individual values within all groups, all values remained within the historical control range (Historical control data corrected body weight gain of dams (period 2012-2017; n = 495); Corrected weight gain (gram): mean = -245; P5 to P95 = -557.3 to 45.4; Corrected weight gain (%): mean = -6; P5 to P95 = -13.8 to 1.3)) and as no dose-related trend was observed.
Body weight and body weight gain at 4.3 and 43 mg/Kg remained in the same range as controls. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Three females (nos. 32, 34 and 35) in the 300 mg/Kg group (sacrificed in extremis on Day 11 or 12 post-coitum before dosing on that day) did not consume food from the start of treatment resulting in a markedly body weight loss (14-17%) and absence of faeces production.
The remaining three females at 300 mg/Kg (nos. 31, 33 and 36; terminated early on Day 12 post-coitum due to animal welfare reasons) were noted with some slight signs of toxicity (body weight loss up to 2-5% and markedly reduced to no food consumption).
At 200 mg/Kg, food consumption was almost absent over the first days of treatment (Days 6-9 post-coitum), followed by a markedly and statistically significantly reduced food consumption over Days 9-12 post-coitum. After correction for body weight, differences of about 88% and 64%, respectively, were noted when compared to the concurrent control group. These changes, both before and after correction of body weight, were statistically significant. Over Days 12-18 post-coitum, food consumption values were slightly lower (not statistically significant) than controls and from Day 18 post-coitum onwards, values remained in the same range as controls.
No toxicologically relevant changes in food consumption were recorded in the 4.3 and 43 mg/Kg groups. The incidentally, but statistically significantly higher mean food consumption (both before and after correction of body weight) at 43 mg/Kg over Days 9-12 post-coitum was related to the relatively high food consumption of one singe female (no. 15) during the first half of the treatment period, and was not considered to be treatment related. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Macroscopic observations at necropsy did not reveal any alterations that were considered to be treatment-related.
All findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rabbits of this age and strain and occurred in absence of a dose-related trend. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Details on results:
- Dose Formulation Analyses:
The concentrations analysed in the formulations of Groups 2 and 3, and Groups 5 and 6 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the Group 1 and 4 formulations. The formulations of Groups 2 and 3, and Groups 5 and 6 were homogeneous (i.e. coefficient of variation ≤ 10%). - Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- The number of implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
As all females at 300 mg/Kg (Group 6) were sacrificed before scheduled necropsy, no developmental data is available for this group. In the 300 mg/Kg group, a mean number of 7.4 normal implantation sites in development per litter was noted, which is within the normal range. - Changes in number of pregnant:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In total, four females were non-pregnant (no. 9 at 4.3 mg/Kg, no. 20 of control Group 4, no. 29 at 200 mg/Kg and no. 35 at 300 mg/Kg). This non-pregnancy rate was not treatment-related as it was equally distributed over the groups and as treatment was initiated from implantation onwards.
- Other effects:
- no effects observed
- Description (incidence and severity):
- The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation. One female at 43 mg/Kg (no. 14) delivered her offspring in the morning of scheduled necropsy (Day 29 post-coitum). She delivered 11 pups, all without developmental malformations. As this occurred in one single female of one of the mid dose groups, it was regarded to be unrelated to treatment.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 43 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: Systemic Toxicity
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean combined (male and female) fetal body weights were 37.3, 39.6 and 37.8 gram for the control, 4.3 and 43 mg/Kg groups, respectively, and 38.4 and 33.0 for the control and 200 mg/Kg group, respectively.
Both male and female fetal body weights were remarkably lower in all litters at 200 mg/Kg when compared to the other groups. The mean value was about 14% lower than the concurrent control group, and was clearly outside the historical control range. - Reduction in number of live offspring:
- not specified
- Description (incidence and severity):
- The numbers of fetuses (litters) available for morphological examination were 60 (6), 50 (5) and 52 (6) in the control (Group 1), 4.3 and 43 mg/Kg/day groups, respectively, and 56 (6) and 45 (5) in the control (Group 4) and 200 mg/Kg group.
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The male:female ratio was unaffected by treatment up to 200 mg/Kg. Mean sex ratios (males:females) were 42:58, 50:50 and 48:52 for the control (Group 1), 4.3 and 43 mg/Kg groups, respectively, and 57:43 and 47:53 for the control (Group 4) and 200 mg/Kg group, respectively.
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- Mean litter sizes were 10.0, 10.0 and 8.7 fetuses/litter for the control (Group 1), 4.3 and 43 mg/Kg groups, respectively, and 11.2 and 9.0 fetuses/litter for the control (Group 4) and 200 mg/Kg group, respectively. The slightly lower mean values at 43 and 200 mg/Kg were not considered to be toxicologically relevant as the changes were marginal and remained in the historical control range. The differences in litter size were related to the high post-implantation loss observed in the 43 and 200 mg/Kg groups.
- External malformations:
- no effects observed
- Description (incidence and severity):
- There were no treatment related effects on external morphology following treatment up to 200 mg/Kg.
There was only one externally malformed fetus (Fetus A006-06) in control Group 1 which was observed to have a distended abdomen, which was confirmed viscerally with ascites.
There were no external variations seen for any fetus in any group. - Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment related effects on skeletal morphology following treatment up to 200 mg/Kg/day.
Two fetuses with skeletal malformations were noted. One fetus of the 4.3 mg/Kg group (A012-04) had a vertebral anomaly with associated rib anomaly. A rib anomaly was also noted for one fetus of control Group 4 (A021-02). As this findings was previously noted in historical control fetuses and occurred in absence of a dose-related trend, it was not considered to be treatment-related.
Skeletal variations occurred at an incidence of 71.0%, 78.1% and 70.3% per litter in control (Group 1), 4.3 and 43 mg/Kg groups, respectively, and 86.8 and 94.4% per litter in the control (Group 4) and 200 mg/Kg group, respectively. All variations that were noted occurred in absence of a dose-related trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, they were not considered treatment-related. - Visceral malformations:
- no effects observed
- Description (incidence and severity):
- There were no treatment related effects on visceral morphology following treatment up to 200 mg/Kg.
Visceral examination revealed two malformed foetuses, both of control Group 1. Fetus A006-6 noted with distended abdomen also had a large heart, and fetus A001-6 was noted with a tetralogy of Fallot.
Visceral variations occurred at incidences of 5.6, 5.0 and 6.1% per litter in the control (Group 1), 4.3 and 43 mg/Kg groups, respectively, and 6.7 and 16.5% per litter in the control (Group 4) and 200 mg/Kg group, respectively. The high incidence in the 200 mg/Kg group was due to one visceral variation (malposition of the left carotid) observed in 7 fetuses of one litter (A027). This finding is the most common variation seen in historical control rabbits, and as it occurred in one single litter, it was not regarded as treatment-related.
All the other variations noted occurred at frequencies that were within the range of available historical control data, and therefore, they were not considered to be treatment-related. - Other effects:
- not specified
- Description (incidence and severity):
- Mean post-implantation loss was 2.8, 4.9 and 13.8% for the control (Group 1), 4.3 and 43 mg/Kg groups, respectively, and 1.3 and 8.3% for the control (Group 4) and 200 mg/Kg group, respectively. The mean value at 43 mg/Kg was outside the historical control range, whereas the value at 200 mg/Kg was around the 95th percentile (i.e. 9.3%).
The changes in post-implantation loss were caused by a high incidence of early and/or late resorptions. In the 43 mg/Kg group, early and late resorptions were observed at incidences of 5.6% and 8.3% respectively (versus 0.0% and 2.8% in the concurrent control group), of which the incidence of late resorptions was outside the historical control range. In the 200 mg/Kg group, no late resorptions were noted, but early resorptions were observed at an incidence of 8.3% (versus 1.3% in the concurrent control group), which was outside the historical control range. None of these changes reached statistical significance, when compared to the concurrent control group. - Key result
- Dose descriptor:
- NOAEL
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Fetal Toxicity
- Remarks on result:
- not determinable
- Remarks:
- A developmental NOAEL could not be established. As based on the data obtained so far, it cannot be concluded whether the increased post-implantation loss at 43 mg/Kg and 200 mg/Kg are related to treatment with the test item, due to the marginal changes and limited group sizes. None of these changes reached statistical significance, when compared to the concurrent control group.
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- not specified
- Conclusions:
- The maternal No Observed Adverse Effect Level (NOAEL) for Phenyl Ethyl Alcohol was determined to be 43 mg/Kg/day.
A fetal NOAEL could not be established since it could not be concluded whether the increased post-implantation loss at 43 mg/Kg and 200 mg/Kg were treatment-related or due to the marginal changes and limited group sizes. On the basis of results observed, the study authors recommended dose levels of 0, 10, 30 and 100 mg/Kg for the definitive GLP prenatal developmental toxicity study in rabbits. The highest dose level of 100 mg/Kg was considered to be the highest suitable dose, since a dose of 200 mg/Kg resulted in severe maternal toxicity. - Executive summary:
A supporting dose range finding study was conducted to determine the potential of Phenyl Ethyl Alcohol (CAS# 60-12-8) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive.
Time-mated female New Zealand White rabbits (6/dose) were exposed to the test material (in a 0.5% aqueous carboxymethyl cellulose + 1.25% Tween 80 vehicle) via oral gavage at dose levels of 0, 4.3, 43, 200 and 300 mg/Kg/day once daily, 7 days a week, from Day 6 to Day 28 post-coitum, inclusive. The dose levels of 4.3 and 43 mg/Kg (Groups 2 and 3) were selected at request of the Sponsor and based on the results of a prenatal developmental toxicity study in rats. As no clear indications of toxicity were observed, additional test material groups were included in this study. The dose levels of these groups (200 and 300 mg/Kg; Groups 5 and 6) were selected based on the results of a tolerability study in non-pregnant rabbits.
Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity. For the F0-generation the parameters evaluated included mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the parameters evaluated for the F1-generation included the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral and skeletal malformations, and developmental variations.
Three out of six females treated at 300 mg/Kg were sacrificed in extremis on Days 11-12 post-coitum, as they had no food consumption from the start of treatment resulting in a marked body weight loss (16-18%) along with absence of faeces production. Additionally, one of these females was observed to be lethargic with uncoordinated movements and a pale and lean appearance. The remaining three females at 300 mg/Kg were noted with some slight signs of toxicity. Because of the severe toxic effects observed in the other half of the animals, these females were terminated early on Day 12 post-coitum due to animal welfare reasons. As all animals at 300 mg/Kg were euthanized before scheduled necropsy, no developmental data was available at this dose level.
At 200 mg/Kg, food consumption was almost absent over the first days of treatment (Days 6-9 post-coitum), followed by a marked reduction in food consumption, especially up to Day 12 post-coitum (relative differences of 64% when compared to controls). Values returned back to normal from Day 18 post-coitum onwards. This resulted in a significant body weight loss (average of 6%) over Days 6-9 post-coitum, followed by a statistically significantly reduced body weight gain over the entire treatment period. Mean body weights were up to 10% lower when compared to controls. Additionally, one female treated at 200 mg/Kg was observed with piloerection.
No toxicologically-related changes were noted in any of the remaining maternal parameters investigated in this study and no maternal toxicity was observed by treatment up to 43 mg/Kg.
Mean post-implantation loss was increased in the 43 and 200 mg/Kg group (about 5 to 6 times higher compared to concurrent controls), caused by a high incidence of early and/or late resorptions. In the 43 mg/Kg group, early and late resorptions were observed at incidences of 5.6% and 8.3% respectively (vs. 0.0% and 2.8% in concurrent controls). In the 200 mg/Kg group, no late resorptions were noted while early resorptions were observed at an incidence of 8.3% (vs. 1.3% in concurrent controls). A dose-related trend was observed and the mean values at 43 and 200 mg/Kg were mostly outside the historical control range (or at the lower end). However, as the differences were marginal and not statistically significant, and as the group size was limited, it cannot be concluded whether these changes are adverse and treatment-related.
Fetal body weights (both males and females) were about 14% lower at 200 mg/Kg than controls. As ossification parameters were unaffected in these fetuses, indicating no growth retardation effects, these changes were regarded to be non-adverse and related to the body weight loss and remarkable lower body weights in the dams of this group.
No treatment-related changes were observed in any of the remaining developmental parameters investigated in the study (i.e. litter size, sex ratio, external, visceral and skeletal malformations, and developmental variations).
Based on the results observed in this dose range finding prenatal developmental toxicity study, the maternal No Observed Adverse Effect Level (NOAEL) for Phenyl Ethyl Alcohol was determined to be 43 mg/Kg.
A fetal NOAEL could not be established since it could not be concluded whether the increased post-implantation loss at 43 mg/Kg and 200 mg/Kg were treatment-related or due to the marginal changes and limited group sizes. On the basis of results observed, the study authors recommended dose levels of 0, 10, 30 and 100 mg/Kg for the definitive GLP prenatal developmental toxicity study in rabbits. The highest dose level of 100 mg/Kg was considered to be the highest suitable dose, since a dose of 200 mg/Kg resulted in severe maternal toxicity.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-07-29 to 2018-10-04
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- 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: Symrise AG (Batch: 208793/A (80100016 (From 29 Jul 2018 until 03 Aug 2018)) and 208793/B (80100036 (from 04 Aug 2018 onwards))
- Expiration date of the lot/batch: 2019-03-22
- Purity test date: Batch: 208793/A (80100016): 2017-06-19 and and 208793/B (80100036): 2018-07-04
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under test conditions: Stable under storage conditions until: 22 March 2019 (expiry date)
- Solubility and stability of the test substance in the solvent/vehicle: Stability in vehicle (0.5% CMC + 1.25% Tween 80) for at least 6 hours at room temperature under normal laboratory light conditions, 8 days in the refrigerator and 3 weeks in the freezer
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear colourless liquid
OTHER SPECIFICS:
Purity/Composition: 99.64%
Molecular formula: C8H10O
pH: 6.0-7.0
Specific gravity / density: 1.0180 (D25/25) - Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: approximately 17-19 weeks old on arrival
- Weight at study initiation: 3130 - 4255 g at the initiation of dosing
- Fasting period before study: Not specified
- Housing: On arrival and following randomization females were housed individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles.
- Diet (e.g. ad libitum): Pelleted diet for rabbits (Global Diet 2030 from Envigo Teklad®, Mucedola, Milanese, Italy) was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during the study period.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles/containers.
- Acclimation period: at least 2 days before the commencement of dosing.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C (actual daily mean temperature during the study period)
- Humidity (%): 54-97% (actual daily mean relative humidity)
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
IN-LIFE DATES: From: 2018-07-27 To: 2018-08-24 (Completion of in-life phase) - Route of administration:
- oral: gavage
- Vehicle:
- other: 0.5% Aqueous carboxymethyl cellulose with 1.25% Tween 80
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a suspension formulated in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial preparations performed at the Test Facility to select a suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment is complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.
- Concentration in vehicle: Group 1 (Control): 0 mg/mL; Group 2: 7.5 mg/mL; Group 3: 25 mg/mL; Group 4: 75 mg/mL
- Amount of vehicle (if gavage): 2 mL/Kg
- Lot/batch no. (if required): Not specified - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analyses were performed by using a validated analytical procedure (Test Facility Study No. 519518).
Concentration Analysis:
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.
Homogeneity Analysis:
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 519518) demonstrated that the test material is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. - Details on mating procedure:
- Time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France). The females arrived on Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were approximately 17-19 weeks old on arrival and weighed between 3130 and 4255 g at the initiation of dosing.
- Duration of treatment / exposure:
- From Day 6 to Day 28 post-coitum, inclusive
- Frequency of treatment:
- Once daily, 7 days a week
- Duration of test:
- Once daily oral gavage 7 days a week from Day 6 to Day 28 post-coitum, inclusive.
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- Group 1 (Control)
- Dose / conc.:
- 15 mg/kg bw/day
- Remarks:
- Group 2
- Dose / conc.:
- 50 mg/kg bw/day
- Remarks:
- Group 3
- Dose / conc.:
- 150 mg/kg bw/day
- Remarks:
- Group 4
- No. of animals per sex per dose:
- 22 females/dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material.
The dose levels have been selected based on the results of a previously performed DRF in pregnant rabbits with Phenyl Ethyl Alcohol (Test Facility Study No. 519517, Sponsor Reference No. 762272), and in an attempt to produce graded responses to the test material:
• In this DRF, the maternal No Observed Adverse Effect Level (NOAEL) was established as being 43 mg/Kg, due to too severe signs of toxicity at 300 mg/Kg (mortality in 3/6 females) and at a lesser extent at 200 mg/Kg (absent or markedly reduced food consumption over Days 6-12 post-coitum, resulting in a 6% body weight loss over Days 6-9 and reduced body weight gain over the entire treatment period).
• A developmental NOAEL could not be established in the DRF due to the marginal (non-statistically significantly) changes and limited group sizes (n = 6 per group). There was a trend towards an increased post-implantation loss at 43 and 200 mg/Kg (about 5 to 6 times higher compared to concurrent controls), caused by a high incidence of early and late resorptions (43 mg/Kg) or early resorptions only (200 mg/Kg).
• Based on the clear toxic effects at 200 mg/Kg in the first days of treatment, which is a critical period of organogenesis, this dose level was considered too high to be used for the main teratology study. Therefore, a dose of 150 mg/Kg was selected as the high dose level for the current study.
- Rationale for animal assignment (if not random): On the day of receipt, animals were assigned to groups by a computer-generated random algorithm according to body weights. Each set of females mated on the same date (i.e. 4 sets) was distributed as evenly as possible over the dose groups with body weights within ± 20% of the mean for each set of animals. - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, in the morning and at the end of the working day (animals were observed for general health/mortality and moribundity)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on Day 6 post-coitum and lasting up to the day prior to necropsy.
BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29 post-coitum
Unscheduled Deaths: If necessary for humane reasons, animals were euthanized as per Test Facility SOPs. These animals were euthanized by intravenous injection of pentobarbital (approx. 1 mL/Kg Euthasol® 20%), underwent necropsy, and specified tissues were retained.
Scheduled Euthanasia:
Animals surviving until scheduled euthanasia were euthanized by intravenous injection of pentobarbital (approx. 1 mL/Kg Euthasol® 20%). No body weight was recorded at necropsy.
Scheduled necropsy was conducted on the following days:
Females surviving to planned necropsy: Day 29 post-coitum (Table 2).
Females with early delivery (no. 28): Within 24 hours of early delivery.
Necropsy: All animals (including animals sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes (Each ovary and uterine horn of all animals was dissected and examined as quickly as possible.
Examinations included
- Gravid uterus weight: Yes (not for animals found dead, sacrificed before planned necropsy or that delivered early)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: a) The number and distribution of live and dead foetuses.; b) The number and distribution of embryo-foetal deaths. - Fetal examinations:
- Live foetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube. Recognizable live foetuses of animals sacrificed before planned necropsy were euthanized by oral administration of sodium pentobarbital.
Foetal Examinations (unscheduled) – F1-Generation
For late resorptions, recognizable foetuses or normal implantations in development of females sacrificed before planned necropsy or that delivered before the day of scheduled necropsy, a gross external examination was performed.
Foetal Examinations (scheduled) – F1-Generation
Litters of females surviving to scheduled necropsy, or that delivered on the day of scheduled necropsy, were subjected to detailed external, visceral and skeletal examinations, as described below.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
- External examinations: Yes: all per litter
Each viable foetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined (not for foetuses of animals sacrificed before planned necropsy). For late resorptions and recognizable foetuses of females euthanized in extremis, a gross external examination was performed. One late resorption with malformation was fixed in 10% buffered formalin.
- Soft tissue examinations: Yes: half per litter
Foetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (Stuckhardt, J.L.; Poppe, S.M. Fresh visceral examination of rat and rabbit fetuses used in teratogenicity testing. Teratogenesis, Carcinogenesis and Mutagenesis,4, 181-188 (1984)). This examination included the heart and major vessels. Foetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (Woo, D.C., Hoar, R.M. Apparent hydronephrosis as a normal aspect of renal development in late gestation of rats: the effect of methyl salicylate. Teratology, 6, 191-196 (1972)).
- Skeletal examinations: Yes: all per litter
All eviscerated foetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson (Dawson, A.B. A note on the staining of skeletons of cleared specimens with Alizarin Red S. Stain Technology.1, 123-124 (1926)). Subsequently, the skeletal examination was done on all foetuses from Groups 1 and 4. Since possible treatment-related effects in the high dose group were observed, skeletal examination was extended to all foetuses from the low and mid dose group. All specimens were archived in glycerin with bronopol as preservative.
Note: A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the foetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
- Head examinations: Yes: all per litter
Heads were removed from approximately one-half of the foetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique (Wilson J.G. - Embryological Considerations in Teratology. In “Teratology: Principles and Techniques”; Wilson, J.G. and Warkany, J., Eds.; The University of Chicago Press: Chicago, IL, 251-277 (1965)). After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the foetuses in each litter of all groups were examined by a mid-coronal slice.
All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons. - Statistics:
- Please see 'Any other information on materials and methods incl. tables' for information on Statistics.
- Indices:
- Maternal Variables
1) Body Weight Gains: Calculated against the body weight on Day 6 post-coitum
2) Corrected Body Weight Gains: Terminal body weight (Day 29 post-coitum) minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
3) Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Reproduction and Developmental Variables
For each group, the following calculations were performed:
1) Pre-implantation loss (%): ((number of corpora lutea – number of implantation sites) / (number of corpora lutea)) x 100
2) Post-implantation loss (%): ((number of implantation sites - number of live foetuses) / (number of implantation sites)) x 100
The foetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of foetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison, calculating the number of affected foetuses as a mean litter proportion on a total group basis, where:
Viable foetuses affected/litter (%): ((number of viable foetuses affected/litter) / (number of viable foetuses/litter)) x 100 - Historical control data:
- Historical Control Data of Fetal Examinations presented in Appendix 3 of the study report (See Tables 13 and 14 below).
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 5 preterm deaths were observed through the study period: 2 controls, 1 animal at 15 mg/Kg and 2 animals at 150 mg/Kg. These deaths were not considered treatment related. All preterm decedents in this study presented with gravid uterus.
In the control group, two animals (nos. 8 and 9) had to be sacrificed for animal welfare reasons on Day 27 post-coitum. They were observed to have severely reduced faeces production (for more than a week), piloerection (for several consecutive days), and/or lean appearance, slight pale skin and hunched posture. Both animals exhibited persistent body weight loss and reduced food consumption from Day 15 post-coitum onwards (up to 8% body weight on Day 27 post-coitum vs start of treatment). No macroscopic findings were noted at necropsy.
At 15 mg/Kg, one animal (no. 28) had to be sacrificed in extremis on Day 21 post-coitum as signs of an early delivery (organic content on the tray) were noted. This female was also observed with severely reduced faeces production (for more than 1 week), piloerection and lean appearance (for 6-7 consecutive days), slight pale skin and hunched posture. Additionally, this animal exhibited a body weight loss of 8% vs start of treatment on Day 21 post-coitum, together with absent food consumption from beginning of treatment onwards. At necropsy, no macroscopic findings were noted in this female. Seven and three normal implantations in development were noted outside and inside the uterus, respectively. Due to the severe signs of maternal toxicity observed, the early delivery in this female was considered to be a consequence of her impaired health status rather than a developmental effect.
At 150 mg/Kg, two animals (nos. 81 and 84) had to be sacrificed in extremis for animal welfare reasons on Day 12 and 27 post-coitum, respectively. Animal no. 81 was observed with transient rales, piloerection, and severely reduced faeces production (for 4 consecutive days), lean appearance (for 2 days), slight pale skin and hunched posture. Additionally, this animal exhibited persistent body weight loss (12% and 14% vs start of dosing on Day 9 and 12 post-coitum, respectively) together with severely reduced food consumption from start of treatment. No macroscopic findings were observed for this animal at necropsy. Animal no. 84 exhibited a severe drop in body weight (~10%) together with almost absent food consumption over Days 24-27 post-coitum. Additionally, severely reduced faeces production and piloerection (for 2 consecutive days), lean appearance, moderate pale skin, and hunched posture were observed. No clinical signs, relevant/persistent body weight loss or reduced food consumption were noted for this animal during the previous days. At necropsy, this female was noted with a gelatinous thymus together with black discoloured left lung and reddish foci in the right lung. Based on these lung findings at necropsy and, as no relevant adverse effects were noted for this animal the previous days, this death might be related to a technical error during the oral gavage procedure.
In surviving animals, reduced faeces production (up to severe) was observed across all the groups, including controls, with a higher persistence/severity at 150 mg/Kg.
At 150 mg/Kg, one animal (no. 78) was observed with transient rales on one single day and one animal (no. 71) was observed with transient lean appearance for 4 consecutive days (over Days 9-12 post-coitum), showing a recovery afterwards. Piloerection was noted in one control female (no. 14) for 2 days, and for several consecutive days (up to 2 weeks) in 3 females at 15 mg/Kg (nos. 32, 36, and 37) and one female at 150 mg/Kg (no. 82). At the incidence observed and in the absence of a dose-related response, these clinical signs in surviving animals were considered not to be test material related.
Red fluid on the manure tray was observed in one control female (no. 17) on a single day and in one animal at 50 mg/Kg (no. 59) for 4 days. In the absence of a dose-related response (occurrence in the control group and in a single mid dose female), this finding was considered unrelated to treatment.
One animal at 150 mg/Kg (no. 67) was noted with a small/short tail. This observation was noted during the animal health check on arrival and, therefore, was considered not to be related to treatment.
Other findings observed included broken upper incisors in one mid dose animal (no. 52), scabs, wounds, scars, alopecia and swelling flews. These findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- There were 5 preterm deaths observed through the study period: 2 controls, 1 animal at 15 mg/Kg and 2 animals at 150 mg/Kg. These deaths were not considered treatment related. All preterm decedents in this study presented with gravid uterus.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- At 15 and 50 mg/Kg, no toxicologically relevant changes in body weight and body weight gain were observed through the study period.
Treatment at 150 mg/Kg resulted in a mean body weight loss of 3% over Days 6-9 post-coitum. Thereafter, mean body weight gain started to recover partially. On average, body weight gain at 150 mg/Kg was statistically significantly lower than control mean from the onset of treatment onwards (not significant only over Days 24-27 post-coitum). This decrease in body weight gain at 150 mg/Kg, resulted in 4-6% lower mean body weights when compared to concurrent controls from start of treatment onwards (reaching statistical significance on Day 29 post-coitum).
At 15 and 50 mg/Kg, mean corrected body weight gain remained in the same range as controls. A slight decrease (non-significant) in mean body weight gain corrected for gravid uterus was observed at 150 mg/Kg (-304.3 vs -137.0 in control). The mean value was within the historical control range . - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- At 15 and 50 mg/Kg, no toxicologically relevant changes in food consumption (before or after allowance for body weight) were observed through the study period.
At 150 mg/Kg, mean food consumption (before or after allowance for body weight) was statistically significantly reduced when compared to concurrent control mean during the first week of treatment, resulting in 53% and 34% lower relative food intake over Days 6-9 and Days 9-12 post-coitum, respectively. Thereafter, mean food intake recovered, showing values comparable to those observed in the concurrent controls from Day 15 post-coitum onwards. - Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
5 preterm deaths were observed through the study period: 2 controls, 1 animal at 15 mg/Kg and 2 animals at 150 mg/Kg. These deaths were not considered treatment related. All preterm decedents in this study presented with gravid uterus.
In the control group, two animals (nos. 8 and 9) had to be sacrificed for animal welfare reasons on Day 27 post-coitum. No macroscopic findings were noted at necropsy.
At 15 mg/Kg, one animal (no. 28) had to be sacrificed in extremis on Day 21 post-coitum as signs of an early delivery (organic content on the tray) were noted. At necropsy, no macroscopic findings were noted in this female.
At 150 mg/Kg, two animals (nos. 81 and 84) had to be sacrificed in extremis for animal welfare reasons on Day 12 and 27 post-coitum, respectively. For animal no. 81, no macroscopic findings were observed for this animal at necropsy. For animal no. 84 necropsy revelated a gelatinous thymus together with black discoloured left lung and reddish foci in the right lung. Based on these lung findings at necropsy and, as no relevant adverse effects were noted for this animal the previous days, this death might be related to a technical error during the oral gavage procedure. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- At 15 mg/Kg, one animal (A028) had to be sacrificed in extremis on Day 21 post-coitum as signs of an early delivery (organic content on the tray) were noted. At necropsy, 7 and 3 normal implantations in development were noted outside and inside the uterus, respectively. Due to the severe signs of maternal toxicity observed in this low dose female, this early delivery was considered to be a consequence of her impaired health status rather than a developmental effect.
The number of pre- and post-implantation sites in the control and treated groups were in the range of normal biological variation. - Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- The number of resorptions in the control and treated groups were in the range of normal biological variation.
Apart from one female at 50 mg/Kg (A059) with only 2 early resorptions, all other pregnant females had litters with viable foetuses. This observation in a mid-dose female was considered a chance finding and unrelated to treatment as it concerned only one female in this group and it was not noted at 150 mg/Kg. - Changes in number of pregnant:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 6 females were observed to be not pregnant: one female at 15 mg/Kg (A042), 4 females at 50 mg/Kg (A045, A061, A062 and A066) and one female at 150 mg/Kg (A069). The incidence of non-pregnancy was considered to be unrelated to treatment as dosing started from implantation (i.e. Day 6 post-coitum) onwards. All the other females in the study (surviving and preterm decedents) were pregnant.
- Other effects:
- no effects observed
- Description (incidence and severity):
- The number of corpora lutea, implantation sites, resorptions, and pre- and post-implantation loss in the control and treated groups were in the range of normal biological variation
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- ca. 50 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- At 150 mg/Kg, a decrease in mean fetal body weight (both males and females) was observed when compared to the control group (males: 35.2g vs 38.5g and females: 34.7g vs 38.3g) with mean values below the 5th percentile of the historical control data (P5=36.9g and 36.3g in males and females, respectively). Although non-statistically significant, mean combined weights was 10% lower than concurrent control mean.
Mean combined (male and female) fetal body weights were 38.7, 38.3, 37.2 and 34.8 gram for the control, 15, 50 and 150 mg/Kg groups, respectively. - Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- The numbers of fetuses (litters) available for foetal morphological examination were 198 (20), 182 (20), 174 (17) and 185 (19) in the control, 15, 50 and 150 mg/kg groups, respectively.
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The male:female ratio was unaffected by treatment up to 150 mg/Kg.
Mean sex ratios (males:females) were 44:56, 49:51, 52:48 and 50:50 for the control, 15, 50 and 150 mg/Kg groups, respectively. - Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related effects on litter size up to 150 mg/Kg.
Mean litter sizes were 9.9, 9.1, 9.7 and 9.7 fetuses/litter for the control, 15, 50 and 150 mg/Kg groups, respectively. - External malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment-related effects on external morphology following treatment up to 150 mg/Kg.
External malformations were observed in 1 (1), 0 (0), 0 (0) and 6 (2) fetuses (litters) in the control, 15, 50 and 150 mg/Kg groups, respectively. In addition, one external malformation was observed in one late resorption at 15 mg/Kg.
Five fetuses from one litter at 150 mg/Kg (A087) exhibited one or multiple malformations: four of these (A087-03, -05, -08 and -12) had an omphalocele and the other one (A087-10) had a short tail. At visceral and skeletal examination, various malformations were also seen among these fetuses, namely an intestine abnormality (A087-08, -10 and -12), malpositioned testis (A087-12) and costal cartilage anomaly (A087-12). The accumulation of all these malformed fetuses in one litter at the high dose level suggests a maternal or genetic origin rather than a toxicological effect. Therefore, these findings were considered not to be related to treatment.
The remaining fetus at 150 mg/Kg affected externally (A076-14) had a cleft palate and carpal flexure. This fetus (A076-14) also exhibited visceral and skeletal abnormalities (i.e. right-sided aortic arch, and skull, vertebral and sternal anomaly). Given the single occurrence (except for vertebral anomaly) of these malformations observed at the high dose level, they were considered chance findings. Moreover, one control foetus (A003-10) also had a carpal flexure, and all findings, except for right-sided aorta, were seen previously among historical control foetuses.
In addition, the malformation bilateral rotated (inwards) hind limbs was observed in one late resorption at 15 mg/Kg (A024-07).
External variations were not observed in this study. - Skeletal malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- A test item-related increase in the number of fetuses that was skeletally malformed occurred at 150 mg/Kg/day.
Skeletal malformations occurred in 1 (1), 1 (1), 0 (0) and 6 (5) fetuses (litters) in the control, 15, 50 and 150 mg/kg/day groups, respectively.
Four foetuses from three litters at 150 mg/kg/day had a vertebral anomaly (fetus A072-04, A076-08 and -14 and A079-04), which led to a mean litter incidence of 2.2% for this malformation at the high dose level. The litter incidence of vertebral anomaly was higher than the concurrent control group (0.4%) and the 95th percentile of the historical control data (P95=1.4%, maximum value =1.5%), and vertebral anomalies in other groups were absent (at 50 mg/kg/day) or occurred in single fetuses (control fetus A003-01 and fetus A044-06 at 15 mg/kg/day). When separating per region of the vertebral column, the litter incidence of vertebral anomaly in the thoracic region remained within the range of the historical control data. However, the litter incidence of vertebral anomaly within the cervical region at 150 mg/kg/day (1.8%) was higher than concurrent control (0.4%) and above the historical control 95th percentile (P95=0.6%, maximum value=1.0%) and, therefore, a relationship to treatment was considered for this finding at this dose level.
Other skeletal malformations noted at the high dose level were: costal cartilage anomaly (fetus A087-12 for which external and visceral malformations were also described), fused skull bones (fetus A078-02) and anomaly of the skull and sternum (fetus A076-14 that was also affected externally and viscerally and was one of the fetuses presenting with vertebral anomaly). Notwithstanding the occurrence of these four different malformations exclusively at 150 mg/kg/day, these were not considered to be related to treatment as they occurred in single fetuses, in combination with external and visceral malformations and/or were previously seen in historical control fetuses.
At 150 mg/kg/day, there was a statistically significant increase in the litter incidence of the skeletal variation 13th full ribs (74.4% vs 43.6% in the control group). The incidence was higher than the 95th percentile of the historical control data (P95=70.9%, maximum value=71.4%) and was accompanied by a higher incidence of caudal shift of pelvic girdle (38.6% vs 15.3% in the control group), which was at the level of the upper historical control limit (P95=34.5%, maximum value=38.5%). Therefore, both skeletal variations were considered to be related to treatment.
Another skeletal variation, supernumerary ossification site of the sternum, was noted in four fetuses from four litters at 150 mg/kg/day. Although the toxicological relevance of this finding was unclear, it was considered to be related to treatment as this skeletal variation was not present in any of the other groups and the mean litter incidence (2.0%) was above the 95th percentile of the historical control data (P95=1.4%, maximum value =1.5%).
In addition, the skeletal variation hyoid body unossified was observed in six fetuses from four litters at 150 mg/kg/day resulting in a mean litter incidence of 3.1%, that was above the historical control range (P95=2.9%) and in the upper limit of the historical control maximum value (3.2%). The higher litter incidence of this ossification-related parameter could be related to the lower mean fetal body weight observed in the high dose group.
All other skeletal variations that were noted in this study occurred at low incidences, in the absence of a dose-related trend and/or at frequencies that were within the range of available historical control data. - Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment-related effects on visceral morphology following treatment up to 150 mg/Kg.
Visceral malformations occurred in 1 (1), 3 (3), 3 (3) and 6 (3) fetuses (litters) in the control, 15, 50 and 150 mg/Kg groups, respectively.
At 150 mg/Kg, besides the visceral malformations observed in litters A076 and A087 listed under external malformations, a small eye was observed in fetus A075-03 and a large heart was noted in fetus A076-08. Fetus A057-05 at 50 mg/Kg also had a small eye (noted during soft tissue cephalic examination). Other visceral malformations at 50 mg/Kg were a large lung cyst in fetus A053-07 and Tetralogy of Fallot in foetus A055-07. Tetralogy of Fallot was also observed in two fetuses at 15 mg/Kg (A026-03 and A035-07) and another foetus at 15 mg/Kg (A033-5) exhibited narrow pulmonary trunk, dilated aortic arch and ventricular septum defect. The viscerally malformed control fetus (A003-09) was observed with a retroesophageal aortic arch. The above visceral malformations in the treated groups were considered unrelated to treatment as they occurred in single fetuses and/or in the absence of a dose-response relationship and/or were previously seen in historical control fetuses.
All visceral variations observed were considered unrelated to treatment as they occurred in the absence of a dose-related trend, at a low incidence, in control fetuses only and/or at frequencies that were within the range of available historical control data. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- ca. 50 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- fetal/pup body weight changes
- skeletal malformations
- Key result
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- skeletal: vertebra
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 150 mg/kg bw/day
- 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:
- not specified
- Conclusions:
- The maternal No Observed Adverse Effect Level (NOAEL) for Phenyl Ethyl Alcohol was determined to be 50 mg/Kg/day, based on effects on reduced maternal body weight gain and food intake observed at 150 mg/Kg/day.
The developmental toxicity NOAEL for Phenyl Ethyl Alcohol was determined to be 50 mg/Kg/day, based on fetal body weight and skeletal malformation observed at 150 mg/Kg/day. - Executive summary:
A key Guideline OECD 414 prenatal developmental toxicity study was conducted to determine the potential of Phenyl Ethyl Alcohol (CAS# 60-12-8) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive.
Time-mated female New Zealand White rabbits (22/dose) were exposed to the test material (in a 0.5% aqueous carboxymethyl cellulose + 1.25% Tween 80 vehicle) via oral gavage at dose levels of 0, 15, 50, 150 mg/Kg/day once daily, 7 days a week, from Day 6 to Day 28 post-coitum, inclusive. The dose levels were based on the results of a previously performed Dose Range Finding (DRF) study in pregnant rabbits with Phenyl Ethyl Alcohol (Test Facility Study No. 519517, Sponsor Reference No. 762272).
For the F0-generation the parameters evaluated included mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents. For the F1-generation, the parameters evaluated included the number of live and dead fetuses, early and late resorptions, total implantations, foetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that formulations of test material in 0.5% Aqueous carboxymethyl cellulose (CMC) with 1.25% Tween 80 were prepared accurately and homogenously.
There were 5 preterm deaths observed through the study period (2 controls, 1 animal at 15 mg/Kg/day and 2 animals at 150 mg/Kg/day). The preterm deaths were not considered to be treatment-related as they occurred in the absence of a dose-related response; mortality was also observed in the control group; and the deaths occurred within the normal limits for rabbits of this strain and age housed in the same conditions as in this study. Additionally, no comparable toxicological effects were observed among the remaining females in these dose groups.
Reduced faeces production (up to severe) was observed in animals across all the groups, including controls, with a higher persistence/severity at 150 mg/Kg. Treatment at 150 mg/Kg/day resulted in a mean body weight loss of 3% over Days 6-9 post-coitum. Although mean body weight gain started to recover partially thereafter, mean body weight gain remained lower than concurrent control mean from the onset of treatment onwards. This decrease in body weight gain resulted in up to 6% lower mean body weights vs control throughout the study period. As complete recovery was not observed and mean foetal body weights were also reduced at this high dose level, the decrease in maternal body weight gain at 150 mg/Kg/day was considered to be adverse.
Although within the range of historical control data, a slight decrease in mean body weight gain corrected for gravid uterus when compared with controls was also observed at 150 mg/Kg. At 150 mg/Kg, mean food consumption was significantly reduced (up to 53%) when compared to concurrent control mean during the first week of treatment, followed by a recovery from Day 15 post-coitum onwards.
At 15 and 50 mg/Kg, no toxicologically relevant changes in body weight, (corrected) body weight gain and food consumption were noted. Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
The number of corpora lutea, implantation sites, resorptions, and pre-implantation loss in the control and treated groups were within the historical control range.
At 150 mg/kg, mean combined foetal body weight was 10% lower vs control and mean values (in both male and females) were below the historical control range. This decrease in foetal body weights was observed at the same dose level at which lower maternal body weight gain from start of treatment until the end of pregnancy was also noted. The reduced fetal body weight was considered to be adverse due to the magnitude of the change indicative of intrauterine/fetal growth retardation.
There were no treatment-related effects on external morphology following treatment up to 150 mg/Kg/day and external variations were not observed in this study. There were no treatment-related effects on visceral morphology following treatment up to 150 mg/Kg/day. All visceral variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, at a low incidence, in control fetuses only and/or at frequencies that were within the range of available historical control data.
At 150 mg/Kg/day, mean combined foetal body weight was 10% lower vs control and mean values (in both males and females) were below the historical control range. This decrease in fetal body weights was observed at the same dose level at which lower maternal body weight gain from start of treatment until the end of pregnancy was also noted. The reduced fetal body weight was considered to be adverse due to the magnitude of the change indicative of intrauterine/foetal growth retardation.
An increase in the litter incidence of vertebral anomaly was observed at 150 mg/Kg/day: four fetuses from three litters at this dose level presented with a vertebral anomaly leading to an increase in the litter incidence of this skeletal malformation when compared to the concurrent control (2.2% vs 0.4%) and historical control data. When the vertebral anomalies were separated by region of the vertebral column, the litter incidence in the thoracic region remained within the range of the historical control data. However, the litter incidence of the vertebral anomaly within the cervical region observed at 150 mg/kg/day was higher than concurrent control (1.8%vs0.4%) and above the upper limit of the historical control range, and therefore a relationship to treatment was considered for this finding at this dose level.
A test item-related increase in the litter incidence of several skeletal variations was also observed at 150 mg/Kg/day.
At 150 mg/Kg/day, there was an increase in the litter incidence of the skeletal variation 13th full ribs when compared to the concurrent control (74.4%vs43.6%) and historical control data, which was accompanied by an increase in the litter incidence of caudal shift of pelvic girdle (38.6%vs15.3% in the control group).
Another skeletal variation, supernumerary ossification site of the sternum, was noted in four fetuses from four litters at 150 mg/Kg/day. Although the toxicological relevance of this finding was unclear, it was considered to be related to treatment as this skeletal variation was not present in any of the other groups and the litter incidence was outside the historical control range.
In addition, the skeletal variation hyoid body unossified was noted in six fetuses from four litters at 150 mg/Kg/day. The higher litter incidence of this ossification-related parameter could be related to the lower mean fetal body weight observed in the high dose group.
No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, external and visceral malformations and developmental variations).
In conclusion, based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for Phenyl Ethyl Alcohol was established as being 50 mg/Kg/day (based on the effects on maternal body weight gain and food intake, fetal body weight and skeletal malformation observed at 150 mg/Kg/day).
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1985
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP-compliant, guideline study not available. Study acceptable for assement.
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- No Guideline available
- GLP compliance:
- yes (incl. QA statement)
- Species:
- rat
- Strain:
- Crj: CD(SD)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:Charles River UK Limited, Manston Road, Margate, Kent
- Age at study initiation: 8 to 9 weeks old
- Weight at study initiation: 170-242 grams
- Housing: individually suspended galvanised metal cages(Bowman R) equipped with solid sides and back, wire mesh front, floor and top.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): S. F Laboratory Diet No.1
- Water (e.g. ad libitum): tap water
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22 °C
- Humidity (%): 53% +/- 11%
- Air changes (per hr):13 air changes per hours
- Photoperiod (hrs dark / hrs light): 12 hours light/ dark cycle (light phase 8.00- 20.00) - Route of administration:
- dermal
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- Before carrying out the test, approximatively 7x5 cm of the intrascapular region of each animal was shaved using electric clippers. Chemical depilation was not applied, rather shaving was repeated during the treatment period. The dermal dose area was protected with a dressing consisting of aluminum foil patch in order to avoid possible ingestion and to minimise the test material evaporation. The dressing covered at least the shaved dorsal area of dose application and was held in place by a medical elastic adhesive bandage (Poroplast).
During the treatment period, the dressing patches was applied immediately after dosing and were not removed until the next morning.
The skin was not washed between applications.
Absorbion of the test material was investigated in "marker" animals which were treated with radiolabelled (C14) phenylethyl alcohol prior to sacrifice. - Details on analytical verification of doses or concentrations:
- The purity of the samples from each of the four different sources was checked by direct injection onto a flame ionization gas chromatograph; a Fluka standard of 99.5% was used as references. Respective percent of purity of samples A, I, N and B were 97.6, 97.5, 99.7 and 99.3.
After analysis, the 4 samples were blended in equal proportions to provide a claculated purity of 98.5% for the blen which was used to treat the animals. - Duration of treatment / exposure:
- Daily dermal application from day 6 of pregnancy to day 15 included.
- Frequency of treatment:
- Once daily
- Duration of test:
- Termination of gestation day 20.
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 143 mg/kg bw/day (nominal)
- Remarks:
- 0.14 ml/kg/day
- Dose / conc.:
- 439 mg/kg bw/day (nominal)
- Remarks:
- 0.43 ml/kg/day
- Dose / conc.:
- 1 428 mg/kg bw/day (nominal)
- Remarks:
- 1.40 ml/kg/day
- No. of animals per sex per dose:
- 25 female - control and 0.43 ml/kg/day
35 females - 0.14 ml/kg/day and 0.140 ml/kg/day - Control animals:
- yes
- Details on study design:
- Dosage volumes were calculated for individual aniimals on Day 6 of the pregnancy and adjusted according to bodyweight on Day 8, 10, 12 and 14.
0.14, 0.43, 1.40 ml/kg/day (nominal conc. (equivalent to 143, 439, 1428 mg/kg bw/d (based on d = 1.0202 g/cm3)))
Exposure: Daily dermal application from day 6 of pregnancy to day 15 included. (Once daily) - Maternal examinations:
- The observations recorded were:
- Sign of reaction to treatment- Daily Observation
- Skin Irritation resulting from treatments- Daily Observation
- Mortalities
- Food Consumption- Daily Observation
- Bodyweights- Observation at day of gestation, day 3, day 6, and alternative day until day 20 of pregnancy
- Laboratory Investigations- Morning of sacrifice
- Hematology
- Biochemistry
- Terminal sacrifice - Ovaries and uterine content:
- The ovaries and uteri were examined immeditely to determine:
- No corpora lutea - Fetal examinations:
- - No and distribution of live young
- No and distribution of embryonic/foetal deaths
- Individual foetal weight
- Foetal abnormalities - Statistics:
- Statistical analysis were performed on litter data, using the litter as basic sample unit and not parametric test as these value rarely follow a normal distribution. Analysis of covariance was used to assess intergroup differences in mean organ weights. The Kruskal -Wallis test was also used to analyse intergroup differences in theresults of the Biochemical and Hematological examination.
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
CLINICAL SIGN
At dosage level of 1.40 ml/kg/day reactions to treatment were observed at day 12 of pregnancy. Those include irritability, a hunched posture, walking on toes, pilo-erection, periorbital staining. Few animals didi not show any signs of reaction.
Administration of 0.43 and 0.14 ml/kg/day did not cause any adverse effect.
MORTALITY
At dosage level of 1.40 ml/kg/day two animals died at day 13 of pregnancy. They showed irritability signs. Autopsy of one animal revealed a slight congestion of the dorsal subcutis.
FOOD CONSUMPTION
At dosage level of 1.40 ml/kg/day food intake decreased significantly.
Administration of 0.43 and 0.14 ml/kg/day did not cause any adverse effect.
BODYWEIGHT
The mean weight gain of animals treated with 1.40 ml/kg/day was markedly retarded.
No difference between treated and control animals was found at dosage levels of 0.43 and 0.14 ml/kg/day did not cause any adverse effect.
HEMATOLOGY and BIOCHEMISTRY
Slight differences in creatinine and GPT were found in animals treated with 0.14 ml/kg/day as well as higher value for white cell count.
ORGAN WEIGHT
No statistically significant differences between test and control.
PREGNANCY RATE
Higher mean implantation rate was found in all groups.
TERMINAL AUTOPSY
No finding in animals treated at dosage level of 1.40 ml/kg/day. - Dose descriptor:
- NOAEL
- Effect level:
- 439 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
LITTER PARAMETERS
1.40 ml/kg/day
Death occurred early in pregnancy. The incidence of embryo-foetal deaths increased significantly. Litter loss: 5/23. Weight and size of litters were also decreased.
0.43 and 0.14 ml/kg/day
No death occurred at these dosage levels. Weight and size were comparable with the controls.
ABNORMALITIES
1.40 ml/kg/day
Morphological changes were observed in 160 of 161 foetuses. Skeleton and soft tissue and specific types of defect were observed in more than 40% of the foetuses: anophthalmia/ microphthalmia, ventricular septal defects, defects/ irregularities affetting thoracic, lumbar and sacrocaudal vertebrae the latter associated with short and or kinky tail, defects of the thoracic ribs and occurrence of cervical ribs.
Soft tissue examination revealed occasional changes in foetuses at 0.14 and 0.43 ml/kg/day. There was not dosage-relationship, but it is considered a threshold.
However, an increased incidence of cervical ribs and defects of thoracic vertebrae was observed. - Dose descriptor:
- LOAEL
- Effect level:
- 143 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: Based on equivocal evidence of increased skeletal variations and delayed skeletal ossification.)Based on equivocal evidence of increased skeletal variations and delayed skeletal ossification.)
- Developmental effects observed:
- not specified
- Conclusions:
- There is a clear evidence of dosage-related increase in the incidence of embryo-foetal toxicity. There was a clear evidence of maternal and embryo-foetal toxicity at the highest dosage level of 1.40 ml/kg/day. The NOAEL for maternal toxicity is considered to be 0.43 ml/kg/day although this is a threshold dosage. A dosage of 0.14 ml/kg/day no adverse effects were observed, therefore it is considered to be the NOEAL for devolopmental toxicity.
- Executive summary:
The study investigated the effect of Phenyl ethyl alcohol in Albino female rats and offspring. The test material was administrated by dermal route at dosage levels of 0.14, 0.40 and 1.40 ml/kg/day from day 6 to day 20 inclusive of pregnancy. The results showed there was a clear evidence of maternal and embryo-foetal toxicity at the highest dosage level of 1.40 ml/kg/day. Skeleton and soft tissue and specific types of defect were observed in more than 40% of the foetuses. Moreover, the incidence of embryo-fetal deaths increased significantly early in pregnancy. Weight and size of litters were also decreased. At 0.43 ml/kg/day there was no evidence of adverse effects on litter values, but there was a dose-dependent increase of morphological changes in fetuses. Finally, the dosage level of 0.14 ml/kg/day did not elicit any adverse effect in both dams and litters.
However, the slight morphological change observed at this dosage cannot be underestimated totally.
The NOAEL for maternal toxicity is considered to be 0.43 ml/kg/day although this is a threshold dosage. A dosage of 0.14 ml/kg/day no adverse effects were observed, therefore it is considered to be the NOEAL for devolopmental toxicity.
Referenceopen allclose all
The test material Phenylethyl alcohol (PEA) alcohol was administrated dermally to 8 female Crj: CD(SD) rats at dose levels of 0, 0.07, 0.14, 0.28, 0.43 and 0.70 ml/kg/day. The dosage-range study was designed to determine:1. wheater cervical ribs, particularly those defined as "vestigial", were the most sensitive indicator of developmental toxicity, 2. wheater this alteration occurred in the absence of both maternal and or other developmental effects, 3. definitive maternal and developmental toxicity no-effect levels. The results of the study shows that no dead rats dead rats occurred in the study. After application of 0.70 ml/kg/day, ptosis for 6 rats of the highest dosage level was observed. Urine- stained abdominal fur was also recorded for 3 rats. In the application area, a grade 1 of erythema and/or desquamation was observed in all rats of all groups. The severity of the effect was dosage-dependent. Skin lesions were observed in 3 rats treated with 0.70 ml/kg/day and 1 treated with 0.28 ml/kg/day. The effects depended on the dermal application of neat PEA. Concerning the foetuses, cervical ribs occurred only at dosage level of 0.70 ml/kg/day.Incompletely ossified ribs occurred at concentration level of 0.043 and 0.70 ml/kg/day. Incompletely ossified sternal centers occurred at all concentration levels. No adverse effects were observed in the controls. Moreover, delayed ossification of the pelvis were observed at 0.07, 0.28, 0.43, 0.70 ml/kg/day. No effects were observed in the controls and no dosage- dependent relationship was demostred.
Ossified metacarpals and ossified hindpaw phalanges per foetus averaged occurred at dosage level of 0.14, 0.28, 0.43, 0.70 ml/kg/day.
Ossified caudal vertebrae per foetus occurred at 0.28, 0.43, 0.70 ml/kg/day, while foetuses with ossified forepaw phalanges occurred only at 0.43, 0.70 ml/kg/day. Other foetal ossifications were observed, but were not dosage-dependent and not significant different from the control groups. In conclusion, the maternal NOAEL is 0.7 ml/kg/day, and the foetal and developmental toxicity NOEAL is 0.07 ml/kg/day.
Table 2. Body Weights (Grams) - Summary |
|||||||
Post Coitum |
|
Group 1 (Control) |
Group 2 (4.3 mg/Kg/day) |
Group 3 (43 mg/Kg/day) |
Group 4 (Control) |
Group 5 (200 mg/Kg/day) |
Group 6 (300 mg/Kg/day) |
Day 2 |
Mean |
3390 |
3442 |
3409 |
3626 |
3554 |
3439 |
St. Dev. |
249.8 |
323.6 |
237.1 |
137.1 |
235.7 |
224.8 |
|
N |
6 |
5 |
6 |
5 |
5 |
5 |
|
|
|||||||
Day 6 |
Mean |
3532 |
3567 |
3512 |
3737 |
3623 |
3494 |
St. Dev. |
251.4 |
264.6 |
229.7 |
144.0 |
267.9 |
252.5 |
|
N |
6 |
5 |
6 |
5 |
5 |
5 |
|
|
|||||||
Day 9 |
Mean |
3577 |
3617 |
3556 |
3816 |
3419* |
3253** |
St. Dev. |
306.8 |
253.2 |
247.2 |
90.9 |
259.9 |
224.4 |
|
N |
6 |
5 |
6 |
5 |
5 |
5 |
|
|
|||||||
Day 12 |
Mean |
3606 |
3683 |
3656 |
3915 |
3509* |
3322** |
St. Dev. |
278.3 |
235.0 |
240.6 |
79.9 |
277.4 |
203.5 |
|
N |
6 |
5 |
6 |
5 |
5 |
4 |
|
|
|||||||
Day 15 |
Mean |
3675 |
3801 |
3711 |
3981 |
3622* |
- |
St. Dev. |
299.5 |
204.4 |
176.0 |
116.0 |
265.8 |
- |
|
N |
6 |
5 |
6 |
5 |
5 |
0 |
|
|
|||||||
Day 18 |
Mean |
3720 |
3846 |
3761 |
4026 |
3633* |
- |
St. Dev. |
295.5 |
233.9 |
217.1 |
115.3 |
253.6 |
- |
|
N |
6 |
5 |
6 |
5 |
5 |
0 |
|
|
|||||||
Day 21 |
Mean |
3710 |
3842 |
3799 |
4048 |
3688* |
- |
St. Dev. |
302.5 |
237.5 |
202.3 |
96.7 |
285.6 |
- |
|
N |
6 |
5 |
6 |
5 |
5 |
0 |
|
|
|||||||
Day 24 |
Mean |
3743 |
3917 |
3782 |
4063 |
3691* |
- |
St. Dev. |
278.7 |
219.2 |
211.5 |
76.7 |
327.0 |
- |
|
N |
6 |
5 |
6 |
5 |
5 |
0 |
|
|
|||||||
Day 27 |
Mean |
3786 |
3978 |
3786 |
4072 |
3683* |
- |
St. Dev. |
282.6 |
223.3 |
247.9 |
117.7 |
345.5 |
- |
|
N |
6 |
5 |
6 |
5 |
5 |
0 |
|
|
|||||||
Day 29 |
Mean |
3821 |
4049 |
3793 |
4105 |
3738 |
- |
St. Dev. |
284.4 |
218.6 |
301.1 |
149.6 |
326.9 |
- |
|
N |
6 |
5 |
6 |
5 |
5 |
0 |
|
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 3. Food Consumption (g/animal/day) - Summary |
|||||||
Post Coitum |
|
Group 1 (Control) |
Group 2 (4.3 mg/Kg/day) |
Group 3 (43 mg/Kg/day) |
Group 4 (Control) |
Group 5 (200 mg/Kg/day) |
Group 6 (300 mg/Kg/day) |
Days 2 - 6 |
Mean |
154 |
148 |
151 |
128 |
108 |
93 |
St. Dev. |
20.5 |
22.5 |
47.2 |
52.7 |
41.0 |
57.5 |
|
N |
6 |
5 |
6 |
5 |
5 |
5 |
|
|
|||||||
Days 6 - 9 |
Mean |
148 |
142 |
141 |
151 |
16** |
11** |
St. Dev. |
31.0 |
26.0 |
39.2 |
18.9 |
17.2 |
12.0 |
|
N |
6 |
5 |
6 |
5 |
5 |
5 |
|
|
|||||||
Days 9 - 12 |
Mean |
130 |
145 |
174* |
152 |
48** |
28** |
St. Dev. |
22.4 |
26.1 |
23.9 |
5.3 |
25.4 |
26.1 |
|
N |
6 |
5 |
6 |
5 |
5 |
5 |
|
|
|||||||
Days 12 - 15 |
Mean |
96 |
126 |
131 |
107 |
78 |
0** |
St. Dev. |
56.8 |
51.0 |
51.6 |
35.1 |
48.8 |
0.5 |
|
N |
6 |
5 |
6 |
5 |
5 |
4 |
|
|
|||||||
Days 15 - 18 |
Mean |
113 |
120 |
127 |
118 |
88 |
|
St. Dev. |
46.5 |
29.8 |
38.6 |
19.8 |
36.3 |
|
|
N |
6 |
5 |
6 |
5 |
5 |
|
|
|
|||||||
Days 18 - 21 |
Mean |
88 |
127 |
125 |
128 |
113 |
|
St. Dev. |
41.8 |
19.1 |
26.6 |
14.0 |
18.4 |
|
|
N |
6 |
5 |
6 |
5 |
5 |
|
|
|
|||||||
Days 21 - 24 |
Mean |
75 |
116 |
87 |
89 |
91 |
|
St. Dev. |
34.9 |
12.9 |
29.6 |
15.2 |
11.7 |
|
|
N |
6 |
5 |
6 |
5 |
5 |
|
|
|
|||||||
Days 24 - 27 |
Mean |
76 |
101 |
63 |
63 |
71 |
|
St. Dev. |
11.0 |
11.5 |
37.6 |
24.8 |
20.5 |
|
|
N |
6 |
5 |
6 |
5 |
5 |
|
|
|
|||||||
Days 27 - 29 |
Mean |
95 |
125 |
81 |
81 |
83 |
|
St. Dev. |
16.7 |
17.4 |
45.0 |
27.2 |
15.1 |
|
|
N |
6 |
5 |
6 |
5 |
5 |
|
|
|
|||||||
Mean of Means |
|
108 |
128 |
120 |
113 |
77 |
33 |
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 4. Summary of Maternal Survival and Pregnancy Status |
||||||||||||
Dose Group |
Group 1 (Control) |
Group 2 (4.3 mg/Kg/day) |
Group 3 (43 mg/Kg/day) |
Group 4 (Control) |
Group 5 (200 mg/Kg/day) |
Group 6 (300 mg/Kg/day) |
||||||
No. |
% |
No. |
% |
No. |
% |
No. |
% |
No. |
% |
No. |
% |
|
Females on Study |
6 |
|
6 |
|
6 |
|
6 |
|
6 |
|
6 |
|
|
||||||||||||
Females that aborted or delivered |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
|
|
|
|
|
|
|
||||||||||||
Females that died |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
Females that aborted |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
Non gravid |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
Gravid |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
|
||||||||||||
Females that were euthanized |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
6 |
100.0 |
Non gravid |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
1 |
16.7 |
Gravid |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
5 |
83.3 |
|
||||||||||||
Females examined at scheduled necropsy |
6 |
100.0 |
6 |
100.0 |
6 |
100.0 |
6 |
100.0 |
6 |
100.0 |
0 |
0.0 |
Non gravid |
0 |
0.0 |
1 |
16.7 |
0 |
0.0 |
1 |
16.7 |
1 |
16.7 |
0 |
0.0 |
Gravid |
6 |
100.0 |
5 |
83.3 |
6 |
100.0 |
5 |
83.3 |
5 |
83.3 |
0 |
0.0 |
With resorptions only |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
With viable fetuses |
6 |
100.0 |
5 |
100.0 |
6 |
100.0 |
5 |
100.0 |
5 |
100.0 |
0 |
0.0 |
|
||||||||||||
Total females Gravid |
6 |
100.0 |
5 |
83.3 |
6 |
100.0 |
5 |
83.3 |
5 |
83.3 |
5 |
83.3 |
Female A014 Delivered Day 29
Table 5. Summary of Fetal Data at Scheduled Necropsy (% per Litter) |
|||||||
Group |
|
Group 1 (Control) |
Group 2 (4.3 mg/Kg/day) |
Group 3 (43 mg/Kg/day) |
Group 4 (Control) |
Group 5 (200 mg/Kg/day) |
Group 6 (300 mg/Kg/day) |
Corpora Lutea |
Mean |
11.0 |
10.6 |
11.0 |
12.4 |
10.8 |
NA |
S.D. |
1.67 |
2.30 |
1.10 |
1.82 |
2.05 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Implantation Sites |
Mean |
10.3 |
10.4 |
10.0 |
11.4 |
9.8 |
NA |
S.D. |
1.51 |
2.07 |
1.10 |
2.30 |
1.30 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Viable Fetuses (%) |
Mean |
97.2 |
95.1 |
86.2 |
98.7 |
91.7 |
NA |
S.D. |
6.82 |
6.83 |
13.84 |
3.00 |
9.14 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Dead Fetuses (%) |
Mean |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
NA |
S.D. |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Early Resorptions (%) |
Mean |
0.0 |
2.9 |
5.6 |
1.3 |
8.3 |
NA |
S.D. |
0.00 |
6.40 |
6.08 |
3.00 |
9.14 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Late Resorptions (%) |
Mean |
2.8 |
2.0 |
8.3 |
0.0 |
0.0 |
NA |
S.D. |
6.82 |
4.47 |
12.88 |
0.00 |
0.00 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Total Resorptions (%) |
Mean |
2.8 |
4.9 |
13.8 |
1.3 |
8.3 |
NA |
S.D. |
6.82 |
6.83 |
13.84 |
3.00 |
9.14 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Pre-Implantation Loss (%) |
Mean |
5.5 |
1.5 |
8.6 |
8.1 |
8.3 |
NA |
S.D. |
10.06 |
3.44 |
10.80 |
11.57 |
8.91 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Post-Implantation Loss (%) |
Mean |
2.8 |
4.9 |
13.8 |
1.3 |
8.3 |
NA |
S.D. |
6.82 |
6.83 |
13.84 |
3.00 |
9.14 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Males (%) |
Mean |
42.3 |
49.9 |
47.8 |
56.7 |
46.6 |
NA |
S.D. |
17.64 |
13.10 |
19.29 |
11.76 |
20.92 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Females (%) |
Mean |
57.7 |
50.1 |
52.2 |
43.3 |
53.4 |
NA |
S.D. |
17.64 |
13.10 |
19.29 |
11.76 |
20.92 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Male Fetal Weights (g) |
Mean |
38.9 |
39.9 |
38.4 |
38.3 |
33.2 |
NA |
S.D. |
4.19 |
4.24 |
6.62 |
5.41 |
5.54 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Female Fetal Weights (g) |
Mean |
36.1 |
39.1 |
36.6 |
38.3 |
32.8 |
NA |
S.D. |
4.43 |
5.53 |
6.17 |
6.99 |
4.00 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
|
|||||||
Combined Fetal Weights (g) |
Mean |
37.3 |
39.6 |
37.8 |
38.4 |
33.0 |
NA |
S.D. |
3.53 |
3.45 |
6.32 |
5.91 |
4.36 |
||
N |
6 |
5 |
6 |
5 |
5 |
||
PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST
FETAL WEIGHTS COMPARED USING DUNNETT'S TEST
None significantly different from control group
NA = NO DAMS SURVIVED TO THE SCHEDULED NECROPSY
Table 6. Summary of Litter Proportions of Malformations (% per Litter) |
|||||||
|
Dose Group: |
Group 1 (Control) |
Group 2 (4.3 mg/Kg/day) |
Group 3 (43 mg/Kg/day) |
Group 4 (Control) |
Group 5 (200 mg/Kg/day) |
Group 6 (300 mg/Kg/day) |
Number of Litters Examined |
|
6 |
5 |
6 |
5 |
5 |
0 |
|
|||||||
Total Malformations |
|
||||||
% per Litter with External Malformations |
Mean |
1.9 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
S.D. |
4.54 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
|
||||||
% per Litter with Soft Tissue Malformations |
Mean |
3.2 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
S.D. |
5.10 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
|
||||||
% per Litter with Skeletal Malformations |
Mean |
0.0 |
1.7 |
0.0 |
1.4 |
0.0 |
0.0 |
S.D. |
0.00 |
3.73 |
0.00 |
3.19 |
0.00 |
0.00 |
|
|
|||||||
Total % per Litter with Malformations |
Mean |
3.2 |
1.7 |
0.0 |
1.4 |
0.0 |
0.0 |
S.D. |
5.10 |
3.73 |
0.00 |
3.19 |
0.00 |
0.00 |
|
None significantly different from control group
Table 7. Summary of Litter Proportions of Variations (% per Litter) |
|||||||
|
Dose Group: |
Group 1 (Control) |
Group 2 (4.3 mg/Kg/day) |
Group 3 (43 mg/Kg/day) |
Group 4 (Control) |
Group 5 (200 mg/Kg/day) |
Group 6 (300 mg/Kg/day) |
Number of Litters Examined |
|
6 |
5 |
6 |
5 |
5 |
0 |
|
|||||||
Total Malformations |
|
||||||
% per Litter with External Variations |
Mean |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
S.D. |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
|
||||||
% per Litter with Soft Tissue Variations |
Mean |
5.6 |
5.0 |
6.1 |
6.7 |
16.5 |
0.0 |
S.D. |
9.30 |
7.45 |
11.01 |
6.51 |
30.39 |
0.00 |
|
|
|
||||||
% per Litter with Skeletal Variations |
Mean |
71.0 |
78.1 |
70.3 |
86.8 |
94.4 |
0.0 |
S.D. |
24.76 |
21.82 |
21.84 |
20.95 |
8.24 |
0.00 |
|
|
|||||||
Total % per Litter with Variations |
Mean |
71.0 |
78.1 |
73.4 |
86.8 |
96.4 |
0.0 |
S.D. |
24.76 |
21.82 |
24.13 |
20.95 |
8.13 |
0.00 |
|
None significantly different from control group
Dose Formulation Analyses
Accuracy:
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test material was detected in the Group 1 formulation.
Homogeneity:
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Table 3. Body Weight Gain (%) – Summary Females |
|||||
Post Coitum |
|
Group 1 Control |
Group 2 15 mg/Kg bw/day |
Group 3 50 mg/Kg bw/day |
Group 4 150 mg/Kg bw/day |
Day 6 |
Mean |
0 |
0 |
0 |
0 |
St. Dev |
0.0 |
0.0 |
0.0 |
0.0 |
|
N |
22 |
21 |
18 |
21 |
|
Day 9 |
Mean |
2 |
2 |
2 |
-3** |
St. Dev |
1.4 |
2.1 |
1.6 |
3.6 |
|
N |
22 |
21 |
18 |
21 |
|
Day 12 |
Mean |
4 |
2 |
3 |
-1** |
St. Dev |
1.6 |
3.1 |
2.2 |
4.1 |
|
N |
22 |
21 |
18 |
21 |
|
Day 15 |
Mean |
5 |
4 |
5 |
1** |
St. Dev |
3.2 |
4.7 |
3.5 |
3.5 |
|
N |
22 |
21 |
18 |
20 |
|
Day 18 |
Mean |
6 |
5 |
6 |
2** |
St. Dev |
2.8 |
4.5 |
3.7 |
4.3 |
|
N |
22 |
21 |
18 |
20 |
|
Day 21 |
Mean |
7 |
5 |
7 |
4* |
St. Dev |
3.1 |
5.1 |
4.3 |
3.6 |
|
N |
22 |
21 |
18 |
20 |
|
Day 24 |
Mean |
8 |
7 |
8 |
5 |
St. Dev |
4.7 |
4.9 |
4.1 |
4.0 |
|
N |
22 |
20 |
18 |
20 |
|
Day 27 |
Mean |
9 |
8 |
9 |
5 |
St. Dev |
5.7 |
5.7 |
4.5 |
5.5 |
|
N |
22 |
20 |
18 |
20 |
|
Day 29 |
Mean |
11 |
9 |
10 |
6** |
St. Dev |
3.9 |
6.5 |
4.7 |
5.6 |
|
N |
20 |
20 |
18 |
19 |
|
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 4. Food Consumption (g/animal/day) – Summary Females |
|||||
Post Coitum |
|
Group 1 Control |
Group 2 15 mg/Kg bw/day |
Group 3 50 mg/Kg bw/day |
Group 4 150 mg/Kg bw/day |
Days 6 - 9 |
Mean |
150 |
137 |
146 |
67** |
St. Dev |
21.2 |
39.1 |
18.8 |
45.6 |
|
N |
22 |
21 |
18 |
21 |
|
Days 9 - 12 |
Mean |
133 |
117 |
137 |
84** |
St. Dev |
16.9 |
50.6 |
24.2 |
37.2 |
|
N |
22 |
21 |
18 |
21 |
|
Days 12 - 15 |
Mean |
86 |
77 |
102 |
70 |
St. Dev |
30.3 |
49.5 |
35.9 |
38.4 |
|
N |
22 |
21 |
18 |
21 |
|
Days 15 - 18 |
Mean |
104 |
95 |
108 |
90 |
St. Dev |
41.9 |
44.5 |
33.5 |
36.8 |
|
N |
22 |
21 |
18 |
20 |
|
Days 18 - 21 |
Mean |
131 |
114 |
128 |
114 |
St. Dev |
38.0 |
48.6 |
29.1 |
30.9 |
|
N |
22 |
21 |
18 |
20 |
|
Days 21 - 24 |
Mean |
99 |
97 |
110 |
95 |
St. Dev |
35.3 |
38.3 |
30.2 |
29.3 |
|
N |
22 |
20 |
18 |
20 |
|
Days 24 - 27 |
Mean |
73 |
79 |
90 |
83 |
St. Dev |
32.4 |
29.9 |
23.6 |
32.9 |
|
N |
22 |
20 |
18 |
20 |
|
Days 27 - 29 |
Mean |
76 |
86 |
85 |
71 |
St. Dev |
39.1 |
31.6 |
25.2 |
35.6 |
|
N |
22 |
20 |
18 |
20 |
|
Mean of Means |
107 |
100 |
113 |
84 |
|
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 5. Relative Food Consumption (g/Kg Body Weight/day) – Summary Females |
|||||
Post Coitum |
|
Group 1 Control |
Group 2 15 mg/Kg bw/day |
Group 3 50 mg/Kg bw/day |
Group 4 150 mg/Kg bw/day |
Days 6 - 9 |
Mean |
40 |
36 |
39 |
19** |
St. Dev |
4.6 |
10.1 |
4.8 |
12.9 |
|
N |
22 |
21 |
18 |
21 |
|
Days 9 - 12 |
Mean |
35 |
31 |
36 |
23** |
St. Dev |
4.3 |
12.9 |
6.5 |
10.0 |
|
N |
22 |
21 |
18 |
21 |
|
Days 12 - 15 |
Mean |
23 |
20 |
27 |
20 |
St. Dev |
8.3 |
12.4 |
9.7 |
10.1 |
|
N |
22 |
21 |
18 |
20 |
|
Days 15 - 18 |
Mean |
27 |
25 |
28 |
24 |
St. Dev |
10.8 |
11.6 |
9.2 |
9.0 |
|
N |
22 |
21 |
18 |
20 |
|
Days 18 - 21 |
Mean |
33 |
29 |
32 |
30 |
St. Dev |
9.8 |
12.1 |
6.8 |
7.2 |
|
N |
22 |
21 |
18 |
20 |
|
Days 21 - 24 |
Mean |
25 |
25 |
28 |
25 |
St. Dev |
9.4 |
9.5 |
7.3 |
7.6 |
|
N |
22 |
20 |
18 |
20 |
|
Days 24 - 27 |
Mean |
18 |
20 |
22 |
22 |
St. Dev |
8.3 |
7.9 |
6.2 |
8.9 |
|
N |
22 |
20 |
18 |
20 |
|
Days 27 - 29 |
Mean |
21 |
21 |
21 |
20 |
St. Dev |
8.1 |
7.9 |
6.3 |
8.8 |
|
N |
20 |
20 |
18 |
19 |
|
Mean of Means |
28 |
26 |
29 |
23 |
|
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 6. Summary of Maternal Survival and Pregnancy Status |
||||||||
Dose Group |
Group 1 Control |
Group 2 15 mg/Kg bw/day |
Group 3 50 mg/Kg bw/day |
Group 4 150 mg/Kg bw/day |
||||
No. |
% |
No. |
% |
No. |
% |
No. |
% |
|
Females on Study |
22 |
|
22 |
|
22 |
|
22 |
|
|
||||||||
Females that aborted |
|
|
|
|
|
|
|
|
or delivered |
0 |
0.0 |
1 |
4.5 |
0 |
0.0 |
0 |
0.0 |
Females that died |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
Females that aborted |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
Non gravid |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
Gravid |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
Females that were euthanized |
2 |
9.1 |
0 |
0.0 |
0 |
0.0 |
2 |
9.1 |
Non gravid |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
Gravid |
2 |
100.0 |
0 |
0.0 |
0 |
0.0 |
2 |
100.0 |
Females examined at Scheduled Necropsy |
29 |
90.0 |
21 |
95.5 |
22 |
100.0 |
20 |
90.9 |
Non gravid |
0 |
0.0 |
1 |
4.8 |
4 |
18.2 |
1 |
5.0 |
Gravid |
20 |
100.0 |
20 |
95.2 |
18 |
81.8 |
19 |
95.0 |
With resorptions only |
0 |
0.0 |
0 |
0.0 |
1 |
5.6 |
0 |
0.0 |
With viable foetuses |
20 |
100.0 |
20 |
100.0 |
17 |
94.4 |
19 |
100.0 |
Total Females Gravid |
22 |
100.0 |
21 |
95.5 |
18 |
81.8 |
21 |
95.5 |
Table 7. Summary of Fetal Data at Scheduled Necropsy |
|||||||||||||
Group |
|
Sex |
Viable Fetuses |
Dead Fetuses |
Resorptions |
Post Implantation Loss |
Implantation Sites |
Corpora Lutea |
Pre Implantation Loss |
Fetal Weight in Grams |
No. of Gravid Females |
||
Male |
Female |
Early |
Late |
||||||||||
1 (Control) |
Total |
86 |
112 |
198 |
0 |
13 |
2 |
15 |
213 |
217 |
4 |
NA |
20 |
Mean |
4.3 |
5.6 |
9.9 |
0.0 |
0.7 |
0.1 |
0.8 |
10.7 |
10.9 |
0.2 |
38.7 |
||
S.D. |
1.72 |
2.01 |
1.77 |
0.00 |
1.09 |
0.45 |
1.12 |
1.87 |
1.87 |
0.41 |
4.36 |
||
|
|||||||||||||
2 (15 mg/Kg bw/day) |
Total |
90 |
92 |
182 |
0 |
7 |
4 |
11 |
193 |
209 |
16 |
NA |
20 |
Mean |
4.5 |
4.6 |
9.1 |
0.0 |
0.4 |
0.2 |
0.6 |
9.7 |
10.5 |
0.8 |
38.3 |
||
S.D. |
1.88 |
1.85 |
2.92 |
0.00 |
0.49 |
0.41 |
0.60 |
3.12 |
2.70 |
1.47 |
5.70 |
||
|
|||||||||||||
3 (50 mg/Kg bw/day) |
Total |
92 |
82 |
174 |
0 |
4 |
2 |
6 |
180 |
184 |
4 |
NA |
18 |
Mean |
5.1 |
4.6 |
9.7 |
0.0 |
0.2 |
0.1 |
0.3 |
10.0 |
10.2 |
0.2 |
37.2 |
||
S.D. |
2.59 |
2.15 |
3.18 |
0.00 |
0.55 |
0.47 |
0.69 |
2.93 |
3.00 |
0.65 |
5.26 |
||
|
|||||||||||||
4 (150 mg/Kg bw/day) |
Total |
93 |
92 |
185 |
0 |
4 |
3 |
7 |
192 |
198 |
6 |
NA |
19 |
Mean |
4.9 |
4.8 |
9.7 |
0.0 |
0.2 |
0.2 |
0.4 |
10.1 |
10.4 |
0.3 |
34.8 |
||
S.D. |
1.88 |
1.50 |
1.94 |
0.00 |
0.42 |
0.50 |
0.60 |
2.13 |
2.06 |
0.75 |
3.79 |
||
None significantly different from control group
NA = Not applicable
Mean Number of Viable Fetuses; Mean Number of Implantation Sites; Mean Number of Corpora Lutea; Fetal Weights compared using Dunnett’s Test
Table 8. Summary of Fetal Data at Scheduled Necropsy (% per Litter) |
|||||
|
Group: |
Group 1 Control |
Group 2 15 mg/Kg bw/day |
Group 3 50 mg/Kg bw/day |
Group 4 150 mg/Kg bw/day |
Corpora Lutea |
Mean |
10.9 |
10.5 |
10.2 |
10.4 |
S.D. |
1.87 |
2.70 |
3.00 |
2.06 |
|
N |
20 |
20 |
18 |
19 |
|
|
|||||
Implantation Sites |
Mean |
10.7 |
9.7 |
10.0 |
10.1 |
S.D. |
1.87 |
3.12 |
2.93 |
2.13 |
|
N |
20 |
20 |
18 |
19 |
|
|
|||||
Viable Fetuses (%) |
Mean |
93.3 |
94.8 |
92.5 |
96.6 |
S.D. |
8.85 |
5.91 |
23.53 |
5.30 |
|
N |
20 |
20 |
18 |
19 |
|
|
|||||
Dead Fetuses (%) |
Mean |
0.0 |
0.0 |
0.0 |
0.0 |
S.D. |
0.00 |
0.00 |
0.00 |
0.00 |
|
N |
20 |
20 |
18 |
19 |
|
|
|||||
Early Resorptions (%) |
Mean |
5.8 |
3.6 |
6.7 |
2.2 |
S.D. |
8.53 |
5.16 |
23.53 |
4.50 |
|
N |
20 |
20 |
18 |
19 |
|
|
|||||
Late Resorptions (%) |
Mean |
0.9 |
1.7 |
0.8 |
1.2 |
S.D. |
4.07 |
3.49 |
3.37 |
3.64 |
|
N |
20 |
20 |
18 |
19 |
|
|
|||||
Total Resorptions (%) |
Mean |
6.7 |
5.2 |
7.5 |
3.4 |
S.D. |
8.85 |
5.91 |
23.53 |
5.30 |
|
N |
20 |
20 |
18 |
19 |
|
|
|||||
Pre Implantation Loss (%) |
Mean |
1.8 |
9.0 |
1.9 |
2.9 |
S.D. |
3.76 |
17.02 |
5.45 |
7.31 |
|
N |
20 |
20 |
18 |
19 |
|
|
|||||
Post Implantation Loss (%) |
Mean |
6.7 |
5.2 |
7.5 |
3.4 |
S.D. |
8.85 |
5.91 |
23.53 |
5.30 |
|
N |
20 |
20 |
18 |
19 |
|
|
|||||
Males (%) |
Mean |
43.8 |
49.5 |
52.1 |
49.7 |
S.D. |
16.25 |
12.08 |
18.20 |
13.72 |
|
N |
20 |
20 |
17 |
19 |
|
|
|||||
Females (%) |
Mean |
56.2 |
50.5 |
47.9 |
50.3 |
S.D. |
16.25 |
12.08 |
18.20 |
13.72 |
|
N |
20 |
20 |
17 |
19 |
|
|
|||||
Male Fetal Weights (g) |
Mean |
38.5 |
39.1 |
38.0 |
35.2 |
S.D. |
5.32 |
6.34 |
5.50 |
4.39 |
|
N |
20 |
20 |
17 |
19 |
|
|
|||||
Female Fetal Weights (g) |
Mean |
38.3 |
37.6 |
36.6 |
34.7 |
S.D. |
4.78 |
5.65 |
5.32 |
4.02 |
|
N |
20 |
20 |
17 |
19 |
|
|
|||||
Combined Fetal Weights (g) |
Mean |
38.7 |
38.3 |
37.2 |
34.8 |
S.D. |
4.36 |
5.70 |
5.26 |
3.79 |
|
N |
20 |
20 |
17 |
19 |
|
Proportional (%) Data Compared using the Mann-Whitney Test
Fetal Weights Compared using Dunnett’s Test
None significantly different from control group
Table 9. Summary of Fetuses and Litters with Malformations (Absolute No.) |
||||||||
Dose Group: |
Fetuses |
Litters |
||||||
Group 1 Control |
Group 2 15 mg/Kg bw/day |
Group 3 50 mg/Kg bw/day |
Group 4 150 mg/Kg bw/day |
Group 1 Control |
Group 2 15 mg/Kg bw/day |
Group 3 50 mg/Kg bw/day |
Group 4 150 mg/Kg bw/day |
|
Number Examined Externally |
198 |
182 |
174 |
185 |
20 |
20 |
17 |
19 |
Carpal and/or Tarsal Flexure |
1 |
0 |
0 |
1 |
1 |
0 |
0 |
1 |
Cleft Palate |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
Trunk Omphalocele |
0 |
0 |
0 |
4 |
0 |
0 |
0 |
1 |
Tail – Absent, Short or Filamentous |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
|
||||||||
Number Examined Viscerally |
198 |
182 |
174 |
185 |
20 |
20 |
17 |
19 |
Teratology or Fallot |
0 |
2 |
1 |
0 |
0 |
2 |
1 |
0 |
Aortic Arch – Retroesophageal |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
Eye(s) Absent and/or Small |
0 |
0 |
1 |
1 |
0 |
0 |
1 |
1 |
Pulmonary Trunk - Narrow |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
Aortic Arch – Dilated |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
Ventricular Septum Defect |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
Viscera Cyst(s) |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
Heart Larger |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
Aortic Arch – Right Sided |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
Intestine – Abnormality |
0 |
0 |
0 |
3 |
0 |
0 |
0 |
1 |
Testis Malpositioned |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
|
||||||||
Number Examined Skeletally |
198 |
182 |
174 |
185 |
20 |
20 |
17 |
19 |
Vertebral Anomaly with or without Rib Anomaly |
1 |
1 |
0 |
4 |
1 |
1 |
0 |
3 |
Costal Cartilage Anomaly |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
Skull Bones Fused |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
Skull Anomaly |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
Sternal Anomaly |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
|
||||||||
Total Number with Malformations |
|
|
|
|
|
|
|
|
External: |
1 |
0 |
0 |
6 |
1 |
0 |
0 |
2 |
Soft Tissue: |
1 |
3 |
3 |
6 |
1 |
3 |
3 |
3 |
Skeletal: |
1 |
1 |
0 |
6 |
1 |
1 |
0 |
5 |
Combined: |
3 |
4 |
3 |
11 |
1 |
4 |
3 |
6 |
Table 10. Summary of Litter Proportions of Malformations (% per Litter) |
|||||
Dose Group: |
|
Group 1 Control |
Group 2 15 mg/Kg bw/day |
Group 3 50 mg/Kg bw/day |
Group 4 150 mg/Kg bw/day |
Number of Litters Examined Externally |
|
20 |
20 |
17 |
19 |
Carpal and/or Tarsal Flexure |
Mean |
0.4 |
0.0 |
0.0 |
0.4 |
S.D. |
1.86 |
0.00 |
0.00 |
1.91 |
|
Cleft Palate |
Mean |
0.0 |
0.0 |
0.0 |
0.4 |
S.D. |
0.00 |
0.00 |
0.00 |
1.91 |
|
Trunk Omphalocele |
Mean |
0.0 |
0.0 |
0.0 |
1.9 |
S.D. |
0.00 |
0.00 |
0.00 |
8.34 |
|
Tail – Absent, Short, or Filamentous |
Mean |
0.0 |
0.0 |
0.0 |
0.5 |
S.D. |
0.00 |
0.00 |
0.00 |
2.09 |
|
|
|||||
Number of Litters Examined Viscerally |
|
20 |
20 |
17 |
19 |
Teratology of Fallot |
Mean |
0.0 |
1.2 |
0.6 |
0.0 |
S.D. |
0.00 |
3.64 |
2.43 |
0.00 |
|
Aortic Arch – Retroesophageal |
Mean |
0.4 |
0.0 |
0.0 |
0.0 |
S.D. |
1.86 |
0.00 |
0.00 |
0.00 |
|
Eye(s) – Absent and/or Small |
Mean |
0.0 |
0.0 |
0.6 |
0.5 |
S.D. |
0.00 |
0.00 |
2.43 |
2.29 |
|
Pulmonary Trunk - Narrow |
Mean |
0.0 |
0.6 |
0.0 |
0.0 |
S.D. |
0.00 |
2.48 |
0.00 |
0.00 |
|
Aortic Arch – Dilated |
Mean |
0.0 |
0.6 |
0.0 |
0.0 |
S.D. |
0.00 |
2.48 |
0.00 |
0.00 |
|
Ventricular Septum Defect |
Mean |
0.0 |
0.6 |
0.0 |
0.0 |
S.D. |
0.00 |
2.48 |
0.00 |
0.00 |
|
Viscera Cyst(s) |
Mean |
0.0 |
0.0 |
0.7 |
0.0 |
S.D. |
0.00 |
0.00 |
3.03 |
0.00 |
|
Heart - Large |
Mean |
0.0 |
0.0 |
0.0 |
0.4 |
S.D. |
0.00 |
0.00 |
0.00 |
1.91 |
|
Aortic Arch – Right Sided |
Mean |
0.0 |
0.0 |
0.0 |
0.4 |
S.D. |
0.00 |
0.00 |
0.00 |
1.91 |
|
Intestine - Abnormality |
Mean |
0.0 |
0.0 |
0.0 |
1.4 |
S.D. |
0.00 |
0.00 |
0.00 |
6.26 |
|
Testis Malpositioned |
Mean |
0.0 |
0.0 |
0.0 |
0.5 |
S.D. |
0.00 |
0.00 |
0.00 |
2.09 |
|
|
|||||
Number of Litters Examined Skeletally |
|
20 |
20 |
17 |
19 |
Vertebral Anomaly with or without Rib anomaly
|
Mean |
0.4 |
0.6 |
0.0 |
2.2 |
S.D. |
1.86 |
2.80 |
0.00 |
5.49 |
|
Costal Cartilage Anomaly |
Mean |
0.0 |
0.0 |
0.0 |
0.5 |
S.D. |
0.00 |
0.00 |
0.00 |
2.09 |
|
Skull Bones – Fused |
Mean |
0.0 |
0.0 |
0.0 |
0.9 |
S.D. |
0.00 |
0.00 |
0.00 |
3.82 |
|
Skull Anomaly |
Mean |
0.0 |
0.0 |
0.0 |
0.4 |
S.D. |
0.00 |
0.00 |
0.00 |
1.91 |
|
Sternal Anomaly |
Mean |
0.0 |
0.0 |
0.0 |
0.4 |
S.D. |
0.00 |
0.00 |
0.00 |
1.91 |
|
|
|||||
Number of Litters Examined |
|
20 |
20 |
17 |
19 |
Total Malformations |
|
|
|
|
|
Percent per Litter with External Malformations |
Mean |
0.4 |
0.0 |
0.0 |
2.8 |
S.D. |
1.86 |
0.00 |
0.00 |
10.50 |
|
Percent per Litter with Soft Tissue Malformations |
Mean |
0.4 |
1.7 |
1.9 |
2.8 |
S.D. |
1.86 |
4.25 |
4.29 |
7.34 |
|
Percent per Litter with Skeletal Malformations |
Mean |
0.4 |
0.6 |
0.0 |
3.6 |
S.D. |
1.86 |
2.80 |
0.00 |
6.47 |
|
|
|||||
Total Percent per Litter with Malformations |
Mean |
1.2 |
2.4 |
1.9 |
6.0 |
S.D. |
5.59 |
4.86 |
4.29 |
11.53 |
|
None significantly different from control group
Table 11. Summary of Fetuses and Litters with Variations (Absolute No.) |
||||||||
Dose Group: |
Fetuses |
Litters |
||||||
Group 1 Control |
Group 2 15 mg/Kg bw/day |
Group 3 50 mg/Kg bw/day |
Group 4 150 mg/Kg bw/day |
Group 1 Control |
Group 2 15 mg/Kg bw/day |
Group 3 50 mg/Kg bw/day |
Group 4 150 mg/Kg bw/day |
|
Number Examined Externally |
198 |
182 |
174 |
185 |
20 |
20 |
17 |
19 |
Number with Findings |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
||||||||
Number Examined Viscerally |
198 |
182 |
174 |
185 |
20 |
20 |
17 |
19 |
Gall Bladder – Absent or small |
4 |
1 |
1 |
2 |
3 |
1 |
1 |
2 |
Ovary - Cyst(s) |
4 |
1 |
1 |
1 |
4 |
1 |
1 |
1 |
Lung – Absent Accessory Lobe |
1 |
4 |
2 |
0 |
1 |
3 |
2 |
0 |
Left Carotid – Originating from Brachiocephalic Trunk |
7 |
4 |
4 |
3 |
6 |
3 |
3 |
2 |
Retrocaval Ureter |
4 |
10 |
1 |
3 |
3 |
6 |
1 |
3 |
Liver – Small Supernumerary Lobe(s) |
1 |
2 |
2 |
0 |
1 |
2 |
2 |
0 |
Gall Bladder – Bilobed |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
Viscera – Cyst(s) |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
Spleen – Pale |
0 |
0 |
0 |
3 |
0 |
0 |
0 |
1 |
Aortic Arch – Supernumerary Artery |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
Renal Papilla(e) – Absent and/or Small |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
|
||||||||
Number Examined Skeletally |
198 |
182 |
174 |
185 |
20 |
20 |
17 |
19 |
13thRudimentary Rib(s) |
5 |
9 |
14 |
5 |
4 |
6 |
9 |
40 |
13thFull Rib(s) |
85 |
81 |
73 |
135 |
16 |
16 |
15 |
18 |
Pelvic Girdle – Caudal Shift |
30 |
10 |
27 |
67 |
8 |
6 |
7 |
14 |
Metacarpal(s) and/or Metatarsal(s) Unossified |
6 |
13 |
6 |
14 |
4 |
6 |
3 |
6 |
Sternebra(e) – Branched |
1 |
2 |
1 |
0 |
1 |
2 |
1 |
0 |
Sternebra (e) - #5 and/or #6 Unossified |
49 |
45 |
62 |
27 |
16 |
17 |
14 |
10 |
7thCervical Full Rib(s) |
1 |
0 |
4 |
2 |
1 |
0 |
2 |
1 |
Sternebra(e) – Malaligned |
8 |
2 |
3 |
9 |
6 |
1 |
2 |
7 |
Pelvic Girdle – Cranial Shift |
1 |
1 |
1 |
0 |
1 |
1 |
1 |
0 |
Hyoid Bone and/or Arch(es) Unossified |
1 |
3 |
2 |
6 |
1 |
3 |
2 |
4 |
Skull – Supernumerary Site |
0 |
1 |
0 |
2 |
0 |
1 |
0 |
2 |
7thCervical Ossification Site(s) |
3 |
1 |
5 |
6 |
2 |
1 |
4 |
4 |
Sternum – Supernumerary Ossification Site |
0 |
0 |
0 |
4 |
0 |
0 |
0 |
4 |
Reduced Ossification of the Skull |
0 |
0 |
0 |
2 |
0 |
0 |
0 |
2 |
Tarsal(s) Unossified |
2 |
2 |
4 |
3 |
2 |
1 |
1 |
2 |
Sternebrae Fused |
1 |
1 |
0 |
2 |
1 |
1 |
0 |
1 |
Caudal Vertebral Anomaly |
0 |
1 |
0 |
1 |
0 |
1 |
0 |
1 |
Skull Bone – Unossified Line |
0 |
1 |
0 |
1 |
0 |
1 |
0 |
1 |
Vertebral Centra – Reduced Ossification |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
Pubis - Unossified |
0 |
1 |
2 |
1 |
0 |
1 |
1 |
1 |
Hyoid Arch(es) Bent |
0 |
2 |
1 |
1 |
0 |
2 |
1 |
1 |
Table 12. Summary of Litter Proportions of Variations (% per Litter) |
|
|||||
Dose Group: |
|
Group 1 Control |
Group 2 15 mg/Kg bw/day |
Group 3 50 mg/Kg bw/day |
Group 4 150 mg/Kg bw/day |
|
Number of Litters Examined Externally |
|
20 |
20 |
17 |
19 |
|
Number of Litters with Findings |
|
0 |
0 |
0 |
0 |
|
|
|
|||||
Number of Litters Examined Viscerally |
|
20 |
20 |
17 |
19 |
|
Gall Bladder – Absent or Small |
Mean |
2.3 |
0.5 |
0.5 |
1.0 |
|
S.D. |
6.03 |
2.24 |
2.02 |
2.87 |
|
|
Ovary – Cyst(s) |
Mean |
2.0 |
0.4 |
0.4 |
0.5 |
|
S.D. |
4.16 |
1.86 |
1.73 |
2.29 |
|
|
Lung – Absent Accessory Lobe |
Mean |
0.5 |
1.8 |
1.2 |
0.0 |
|
S.D. |
2.24 |
4.38 |
3.51 |
0.00 |
|
|
Left Carotid – Originating from Brachiocephalic Trunk |
Mean |
3.2 |
1.9 |
2.2 |
1.5 |
|
S.D. |
5.13 |
4.76 |
5.02 |
4.65 |
|
|
Retrocaval Ureter |
Mean |
2.2 |
5.4 |
0.5 |
1.5 |
|
S.D. |
5.55 |
10.17 |
2.20 |
3.55 |
|
|
Liver – Small Supernumerary Lobe(s) |
Mean |
0.4 |
0.9 |
1.2 |
0.0 |
|
S.D. |
1.86 |
2.83 |
3.51 |
0.00 |
|
|
Gall Bladder – Bilobed |
Mean |
0.0 |
0.0 |
0.0 |
0.4 |
|
S.D. |
0.00 |
0.00 |
0.00 |
1.91 |
|
|
Viscera – Cyst(s) |
Mean |
0.5 |
0.0 |
0.0 |
0.0 |
|
S.D. |
2.24 |
0.00 |
0.00 |
0.00 |
|
|
Spleen – Pale |
Mean |
0.0 |
0.0 |
0.0 |
1.3 |
|
S.D. |
0.00 |
0.00 |
0.00 |
5.74 |
|
|
Aortic Arch – Supernumerary Artery |
Mean |
0.0 |
0.6 |
0.0 |
0.0 |
|
S.D. |
0.00 |
2.48 |
0.00 |
0.00 |
|
|
Renal Papilla(e) – Absent and/or Small |
Mean |
0.0 |
0.4 |
0.0 |
0.0 |
|
S.D. |
0.00 |
1.86 |
0.00 |
0.00 |
|
|
|
|
|||||
Number of Litters Examined Skeletally |
|
20 |
20 |
17 |
19 |
|
13thRudimentary Rib(s) |
Mean |
2.5 |
4.4 |
8.0 |
2.5 |
|
S.D. |
6.15 |
8.28 |
10.31 |
5.67 |
|
|
13thFull Rib(s) |
Mean |
43.6 |
45.7 |
38.5 |
74.4** |
|
S.D. |
37.67 |
37.48 |
36.85 |
30.04 |
|
|
Pelvic Girdle – Caudal Shift |
Mean |
15.3 |
7.1 |
13.6 |
38.6* |
|
S.D. |
24.14 |
13.45 |
25.45 |
32.11 |
|
|
Metacarpal(s) and/or Metatarsal(s) Unossified |
Mean |
2.4 |
5.4 |
3.4 |
6.7 |
|
S.D. |
5.78 |
11.98 |
8.33 |
12.68 |
|
|
Sternebra(e) – Branched |
Mean |
0.4 |
1.1 |
0.6 |
0.0 |
|
S.D. |
1.86 |
3.42 |
2.43 |
0.00 |
|
|
Sternebra (e) - #5 and/or #6 Unossified |
Mean0 |
26.6 |
25.9 |
35.3 |
14.8 |
|
S.D. |
26.30 |
21.80 |
27.24 |
17.08 |
|
|
7thCervical Full Rib(s) |
Mean |
0.5 |
0.0 |
2.7 |
1.1 |
|
S.D. |
2.24 |
0.00 |
9.22 |
4.59 |
|
|
Sternebra(e) – Malaligned |
Mean |
3.8 |
1.1 |
1.4 |
4.8 |
|
S.D. |
6.62 |
4.97 |
4.21 |
7.43 |
|
|
Pelvic Girdle – Cranial Shift |
Mean |
0.5 |
0.5 |
0.5 |
0.0 |
|
S.D. |
2.24 |
2.03 |
2.02 |
0.00 |
|
|
Hyoid Bone and/or Arch(es) Unossified |
Mean |
0.5 |
1.6 |
0.9 |
3.1 |
|
S.D. |
2.24 |
3.90 |
2.66 |
6.92 |
|
|
Skull – Supernumerary Site |
Mean |
0.0 |
0.4 |
0.0 |
1.0 |
|
S.D. |
0.00 |
1.60 |
0.00 |
2.90 |
|
|
7thCervical Ossification Site(s) |
Mean |
2.0 |
0.6 |
3.1 |
3.4 |
|
S.D. |
6.73 |
2.80 |
6.76 |
7.42 |
|
|
Sternum – Supernumerary Ossification Site |
Mean |
0.0 |
0.0 |
0.0 |
2.0* |
|
S.D. |
0.00 |
0.00 |
0.00 |
4.11 |
|
|
Reduced Ossification of the Skull |
Mean |
0.0 |
0.0 |
0.0 |
1.0 |
|
S.D. |
0.00 |
0.00 |
0.00 |
2.90 |
|
|
Tarsal(s) Unossified |
Mean |
0.8 |
0.8 |
2.0 |
1.3 |
|
S.D. |
2.59 |
3.73 |
8.08 |
4.18 |
|
|
Sternebrae Fused |
Mean |
0.7 |
0.6 |
0.0 |
1.2 |
|
S.D. |
3.19 |
2.48 |
0.00 |
5.10 |
|
|
Caudal Vertebral Anomaly |
Mean |
0.0 |
1.3 |
0.0 |
0.4 |
|
S.D. |
0.00 |
5.59 |
0.00 |
1.76 |
|
|
Skull Bone – Unossified Line |
Mean |
0.0 |
0.5 |
0.0 |
0.5 |
|
S.D. |
0.00 |
2.24 |
0.00 |
2.29 |
|
|
Vertebral Centra – Reduced Ossification |
Mean |
0.0 |
0.0 |
0.0 |
0.4 |
|
S.D. |
0.00 |
0.00 |
0.00 |
1.91 |
|
|
Pubis - Unossified |
Mean |
0.0 |
0.4 |
1.0 |
0.4 |
|
S.D. |
0.00 |
1.86 |
4.04 |
1.91 |
|
|
Hyoid Arch(es) Bent |
Mean |
0.0 |
0.9 |
0.7 |
0.4 |
|
S.D. |
0.00 |
2.88 |
2.69 |
1.91 |
|
|
|
||||||
Number of Litters Examined |
|
20 |
20 |
17 |
19 |
|
Total Variations |
|
|
|
|
|
|
Percent per Litter with External Variations |
Mean |
0.0 |
0.0 |
0.0 |
0.0 |
|
S.D. |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
Percent per Litter with Soft Tissue Variations |
Mean |
11.1 |
11.1 |
6.1 |
6.2 |
|
S.D. |
7.74 |
12.92 |
8.76 |
11.62 |
|
|
Percent per Litter with Skeletal Variations |
Mean |
70.3 |
69.0 |
72.9 |
86.6 |
|
S.D. |
27.27 |
26.69 |
27.49 |
16.31 |
|
|
|
|
|||||
Total Percent per Litter with Variations |
Mean |
72.4 |
71.9 |
73.9 |
86.6 |
|
S.D. |
25.75 |
24.75 |
27.27 |
16.32 |
|
|
None significantly different from control group
Table 13. Historical Control Data: Albino, New Zealand White Rabbit (Malformations) |
|||||||||
No. of Studies: 26 |
|||||||||
Total No. Fetuses (Litters) Examined Externally: 4513 (495) |
|||||||||
Total No. Fetuses (Litters) Examined Viscerally: 4513 (495) |
|||||||||
Malformations |
Mean of Study Means (% per Litter Basis) |
Median |
Min |
Max |
P5 |
P95 |
Summary Incidence (Total No. Affected) |
||
Mean |
SD |
Fetuses |
Litters |
||||||
TOTAL EXTERNAL MALFORMATIONS |
|
|
|
|
|
|
|
16 |
13 |
TOTAL VISCERAL MALFORMATIONS |
|
|
|
|
|
|
|
73 |
62 |
TOTAL SKELETAL MALFORMATIONS |
|
|
|
|
|
|
|
62 |
56 |
TOTAL MALFORMATIONS |
|
|
|
|
|
|
|
135 |
112 |
|
|||||||||
EXTERNAL |
|
|
|
|
|
|
|
|
|
Abdomen- Distended |
0.3 |
0.42 |
0.3 |
0.0 |
0.6 |
0.0 |
0.4 |
1 |
1 |
Brachydactyly |
0.0 |
0.17 |
0.0 |
0.0 |
0.7 |
0.0 |
0.6 |
2 |
2 |
Carpal and/or Tarsal Flexure |
0.2 |
0.35 |
0.0 |
0.0 |
1.1 |
0.0 |
1.1 |
9 |
6 |
Cleft Lip |
0.0 |
0.11 |
0.0 |
0.0 |
0.4 |
0.0 |
0.3 |
1 |
1 |
Cleft Palate |
0.1 |
0.23 |
0.0 |
0.0 |
1.1 |
0.0 |
0.9 |
4 |
2 |
Exencephaly |
0.0 |
0.10 |
0.0 |
0.0 |
0.5 |
0.0 |
0.3 |
1 |
1 |
Ectrodactyly |
0.0 |
0.15 |
0.0 |
0.0 |
0.6 |
0.0 |
0.6 |
2 |
2 |
Eye(s) - Open |
0.0 |
0.12 |
0.0 |
0.0 |
0.5 |
0.0 |
0.5 |
2 |
2 |
Limb(s)- Short |
0.1 |
0.20 |
0.0 |
0.0 |
0.7 |
0.0 |
0.5 |
1 |
1 |
Omphalocele |
0.0 |
0.14 |
0.0 |
0.0 |
0.6 |
0.0 |
0.5 |
2 |
2 |
Subcutaneous Edema - Generalized |
0.0 |
0.08 |
0.0 |
0.0 |
0.4 |
0.0 |
0.3 |
1 |
1 |
|
|||||||||
VISCERAL |
|
|
|
|
|
|
|
|
|
Abdomen- Ascites |
0.1 |
0.16 |
0.0 |
0.0 |
0.5 |
0.0 |
0.3 |
1 |
1 |
Aortic Arch- Dilated |
0.1 |
0.23 |
0.0 |
0.0 |
0.9 |
0.0 |
0.8 |
6 |
5 |
Aortic Arch- Interrupted |
0.0 |
0.08 |
0.0 |
0.0 |
0.4 |
0.0 |
0.3 |
1 |
1 |
Aortic Arch- Narrow |
0.3 |
0.35 |
00.3 |
0.0 |
0.5 |
0.0 |
0.3 |
1 |
1 |
Atrial Septum- Defect |
0.1 |
0.17 |
0.0 |
0.0 |
0.6 |
0.0 |
0.6 |
4 |
3 |
Atrioventricular Valve- Absent |
0.3 |
0.42 |
0.3 |
0.0 |
0.6 |
0.0 |
0.4 |
1 |
1 |
Atrium- Large |
0.3 |
0.42 |
0.3 |
0.0 |
0.6 |
0.0 |
0.4 |
1 |
1 |
Diaphragm- Cyst |
0.0 |
0.10 |
0.0 |
0.0 |
0.5 |
0.0 |
0.3 |
1 |
1 |
Diaphragm- Hernia |
0.3 |
0.42 |
0.3 |
0.0 |
0.6 |
0.0 |
0.4 |
1 |
1 |
Eye(s)- Absent And/Or Small |
0.0 |
0.10 |
0.0 |
0.0 |
0.5 |
0.0 |
0.3 |
1 |
1 |
Eye(s)- Retina Folded |
0.1 |
0.25 |
0.0 |
0.0 |
1.3 |
0.0 |
0.8 |
1 |
1 |
Fistula |
0.0 |
0.08 |
0.0 |
0.0 |
0.4 |
0.0 |
0.3 |
1 |
1 |
Heart- Large |
0.1 |
0.16 |
0.0 |
0.0 |
0.5 |
0.0 |
0.3 |
1 |
1 |
Hydrocephaly- Internal |
0.1 |
0.23 |
0.0 |
0.0 |
0.8 |
0.0 |
0.8 |
4 |
3 |
Intestine- Large |
0.2 |
0.30 |
0.0 |
0.0 |
0.6 |
0.0 |
0.4 |
1 |
1 |
Kidney(s)- Fused |
0.0 |
0.14 |
0.0 |
0.0 |
0.7 |
0.0 |
0.5 |
1 |
1 |
Kidney(s)- Malpositioned |
0.1 |
0.18 |
0.0 |
0.0 |
0.7 |
0.0 |
0.7 |
2 |
2 |
Liver- Abnormal Lobation |
0.0 |
0.14 |
0.0 |
0.0 |
0.6 |
0.0 |
0.5 |
2 |
2 |
Lung- Abnormal Lobation |
0.1 |
0.20 |
0.0 |
0.0 |
0.7 |
0.0 |
0.7 |
3 |
3 |
Lung- Absent Lung Lobe(s) |
0.5 |
0.59 |
0.6 |
0.0 |
1.7 |
0.0 |
1.7 |
29 |
26 |
Lung- Cyst |
0.0 |
0.08 |
0.0 |
0.0 |
0.4 |
0.0 |
0.3 |
1 |
1 |
Lung- Small Lung Lobe(s) |
0.0 |
0.08 |
0.0 |
0.0 |
0.4 |
0.0 |
0.3 |
1 |
1 |
Pulmonary Trunk- Atretic |
0.0 |
0.14 |
0.0 |
0.0 |
0.6 |
0.0 |
0.5 |
3 |
2 |
Pulmonary Trunk- Narrow |
0.1 |
0.20 |
0.0 |
0.0 |
0.9 |
0.0 |
0.8 |
3 |
3 |
Subclavian (Right)- Originating from the Pulmonary Trunk |
0.1 |
0.31 |
0.0 |
0.0 |
0.7 |
0.0 |
0.5 |
1 |
1 |
Testis- Malpositioned |
0.2 |
0.52 |
0.0 |
0.0 |
2.0 |
0.0 |
1.9 |
10 |
8 |
Tetralogy of Fallot |
0.2 |
0.26 |
0.0 |
0.0 |
0.8 |
0.0 |
0.7 |
8 |
7 |
Truncus Arteriosus- Persistent |
0.0 |
0.15 |
0.0 |
0.0 |
0.6 |
0.0 |
0.6 |
2 |
2 |
Ventricular Septum- Defect |
0.1 |
0.18 |
0.0 |
0.0 |
0.5 |
0.0 |
0.5 |
5 |
5 |
|
|||||||||
SKELETAL |
|
|
|
|
|
|
|
|
|
Caudal Vertebral Anomaly |
0.1 |
0.23 |
0.0 |
0.0 |
0.8 |
0.0 |
0.8 |
4 |
4 |
Costal Cartilage Anomaly |
0.1 |
0.18 |
0.0 |
0.0 |
0.7 |
0.0 |
0.7 |
2 |
2 |
Limb Bones- Bent |
0.0 |
0.17 |
0.0 |
0.0 |
0.7 |
0.0 |
0.6 |
2 |
2 |
Rib Anomaly |
0.0 |
0.15 |
0.0 |
0.0 |
0.6 |
0.0 |
0.6 |
2 |
2 |
Skull- Anomaly |
0.1 |
0.17 |
0.0 |
0.0 |
0.7 |
0.0 |
0.6 |
3 |
3 |
Skull- Sutural Bone |
0.0 |
0.10 |
0.0 |
0.0 |
0.5 |
0.0 |
0.3 |
1 |
1 |
Sternal Anomaly |
0.0 |
0.16 |
0.0 |
0.0 |
0.8 |
0.0 |
0.5 |
1 |
1 |
Sternoschisis |
0.0 |
0.14 |
0.0 |
0.0 |
0.7 |
0.0 |
0.5 |
1 |
1 |
Vertebral Anomaly With or Without Associated Rib Anomaly |
0.4 |
0.50 |
0.0 |
0.0 |
1.5 |
0.0 |
1.4 |
17 |
15 |
Table 14. Historical Control Data: Albino, New Zealand White Rabbit (Variations) |
|||||||||
No. of Studies: 26 |
|||||||||
Total No. Fetuses (Litters) Examined Externally: 4513 (495) |
|||||||||
Total No. Fetuses (Litters) Examined Viscerally: 4513 (495) |
|||||||||
Variations |
Mean of Study Means (% per Litter Basis) |
Median |
Min |
Max |
P5 |
P95 |
Summary Incidence (Total No. Affected) |
||
Mean |
SD |
Fetuses |
Litters |
||||||
TOTAL EXTERNAL MALFORMATIONS |
|
|
|
|
|
|
|
16 |
13 |
TOTAL VISCERAL MALFORMATIONS |
|
|
|
|
|
|
|
73 |
62 |
TOTAL SKELETAL MALFORMATIONS |
|
|
|
|
|
|
|
62 |
56 |
TOTAL MALFORMATIONS |
|
|
|
|
|
|
|
135 |
112 |
|
|||||||||
EXTERNAL |
|
|
|
|
|
|
|
|
|
None Observed |
|
|
|
|
|
|
|
|
|
|
|||||||||
VISCERAL |
|
|
|
|
|
|
|
|
|
Adrenal- Supernumerary |
0.1 |
0.21 |
0.0 |
0.0 |
0.9 |
0.0 |
0.8 |
2 |
2 |
Aortic Arch- Supernumerary Artery |
1.3 |
1.63 |
0.7 |
0.0 |
6.1 |
0.0 |
5.6 |
56 |
45 |
Carotid (Left)- Originating from Brachiocephalic Trunk |
2.6 |
2.90 |
1.7 |
0.0 |
111.4 |
0.0 |
10.6 |
118 |
72 |
Eye- Hemorrhagic Ring around the Iris |
0.1 |
0.35 |
0.0 |
0.0 |
1.8 |
0.0 |
1.2 |
1 |
1 |
Gallbladder- Absent or Small |
0.8 |
0.68 |
0.6 |
0.0 |
2.4 |
0.0 |
2.3 |
37 |
34 |
Liver- Appendix |
0.2 |
0.40 |
0.0 |
0.0 |
1.5 |
0.0 |
1.4 |
9 |
8 |
Liver- Cyst |
0.1 |
0.29 |
0.0 |
0.0 |
1.3 |
0.0 |
1.1 |
5 |
4 |
Liver- Discolored |
0.1 |
0.18 |
0.0 |
0.0 |
0.6 |
0.0 |
0.6 |
3 |
3 |
Liver- Small Supernumerary Lobe(s) |
0.3 |
0.68 |
0.0 |
0.0 |
2.7 |
0.0 |
2.4 |
11 |
9 |
Lung- Absent Accessory Lobe |
0.7 |
1.27 |
0.0 |
0.0 |
2.2 |
@ |
@ |
4 |
2 |
Lung- Discolored |
0.0 |
0.12 |
0.0 |
0.0 |
0.6 |
0.0 |
0.4 |
1 |
1 |
Ovary- Cyst |
0.3 |
0.34 |
0.0 |
0.0 |
1.3 |
0.0 |
1.1 |
12 |
12 |
Renal Papilla(e)- Absent and/or Small |
0.1 |
0.43 |
0.0 |
0.0 |
2.1 |
0.0 |
1.5 |
4 |
4 |
Spleen- Constricted |
0.4 |
0.75 |
0.0 |
0.0 |
3.1 |
0.0 |
2.6 |
17 |
15 |
Spleen- Pale |
0.1 |
0.28 |
0.0 |
0.0 |
1.3 |
0.0 |
1.1 |
3 |
2 |
Spleen- Small |
0.0 |
0.14 |
0.0 |
0.0 |
0.7 |
0.0 |
0.5 |
1 |
1 |
Spleen- Supernumerary |
0.1 |
0.29 |
0.0 |
0.0 |
1.2 |
0.0 |
1.0 |
5 |
4 |
Subclavian (Right)- Retroesophageal |
0.1 |
0.35 |
0.0 |
0.0 |
1.4 |
0.0 |
1.3 |
6 |
6 |
Testis- Cyst(s) |
0.1 |
0.29 |
0.0 |
0.0 |
1.1 |
0.0 |
1.0 |
7 |
6 |
Thymus- Partially Undescended Horn(s) |
0.9 |
0.98 |
0.5 |
0.0 |
3.0 |
0.0 |
3.0 |
44 |
34 |
Thyroid(s)- Large |
0.0 |
0.10 |
0.0 |
0.0 |
0.5 |
0.0 |
0.3 |
1 |
1 |
Ureter(s)- Convoluted |
0.0 |
0.08 |
0.0 |
0.0 |
0.4 |
0.0 |
0.3 |
1 |
1 |
Ureter(s)- Dilated |
0.0 |
0.14 |
0.0 |
0.0 |
0.5 |
0.0 |
0.5 |
2 |
2 |
Ureter(s)- Retrocaval |
2.1 |
1.52 |
1.5 |
0.0 |
6.3 |
0.0 |
5.5 |
88 |
64 |
Viscera- Attachment(s) |
0.0 |
0.14 |
0.0 |
0.0 |
0.5 |
0.0 |
0.5 |
2 |
2 |
|
|||||||||
SKELETAL |
|
|
|
|
|
|
|
|
|
7th Cervical Full Rib(s) |
0.2 |
0.43 |
0.0 |
0.0 |
1.5 |
0.0 |
1.4 |
11 |
9 |
7th Cervical Ossification Site(s) |
2.3 |
4.10 |
1.0 |
0.0 |
19.9 |
0.0 |
15.1 |
87 |
50 |
13th Full Rib(s) |
48.1 |
12.38 |
47.9 |
26.8 |
71.4 |
27.2 |
70.9 |
2225 |
455 |
13th Rudimentary Rib(s) |
10.5 |
3.05 |
10.5 |
3.8 |
17.3 |
4.2 |
17.1 |
468 |
289 |
Caudal Vertebral Anomaly |
0.5 |
0.50 |
0.5 |
0.0 |
1.7 |
0.0 |
1.7 |
25 |
25 |
Hyoid Arch(es) Bent |
0.3 |
0.46 |
0.0 |
0.0 |
1.4 |
0.0 |
1.4 |
11 |
11 |
Hyoid Body and/or Arches Unossified |
0.7 |
0.86 |
0.4 |
0.0 |
3.2 |
0.0 |
2.9 |
38 |
30 |
Metacarpal(s) and/or Metatarsal(s) Unossified |
4.2 |
2.60 |
4.2 |
0.0 |
10.1 |
0.0 |
9.4 |
226 |
106 |
Pelvic Girdle- Caudal Shift |
16.5 |
7.60 |
16.5 |
2.5 |
38.5 |
2.8 |
34.5 |
786 |
277 |
Pelvic Girdle- Cranial Shift |
0.2 |
0.43 |
0.0 |
0.0 |
1.9 |
0.0 |
1.6 |
6 |
6 |
Pubis- Unossified |
0.2 |
0.29 |
0.0 |
0.0 |
0.8 |
0.0 |
0.8 |
12 |
10 |
Rib(s)- Nodulated |
0.1 |
0.24 |
0.0 |
0.0 |
0.8 |
0.0 |
0.8 |
4 |
4 |
Skull Bones- Fused |
0.0 |
0.15 |
0.0 |
0.0 |
0.6 |
0.0 |
0.6 |
2 |
2 |
Skull Bone(s)- Split |
0.0 |
0.10 |
0.0 |
0.0 |
0.4 |
0.0 |
0.4 |
2 |
2 |
Skull Bone- Unossified Line |
0.4 |
0.53 |
0.2 |
0.0 |
2.3 |
0.0 |
1.9 |
19 |
18 |
Skull- Reduced Ossification |
0.1 |
0.25 |
0.0 |
0.0 |
0.8 |
0.0 |
0.8 |
7 |
5 |
Skull- Supernumerary Site |
0.9 |
0.95 |
0.7 |
0.0 |
3.6 |
0.0 |
3.3 |
41 |
32 |
Sternebra(e) #1.#2.#3 and/or #4 Unossified |
0.0 |
0.08 |
0.0 |
0.0 |
0.4 |
0.0 |
0.3 |
1 |
1 |
Sternebra(e) #5 and/or #6 Unossified |
19.0 |
5.14 |
19.0 |
3.0 |
27.5 |
6.1 |
27.0 |
861 |
310 |
Sternebra(e)- Branched |
0.1 |
0.34 |
0.0 |
0.0 |
1.2 |
0.0 |
1.1 |
5 |
5 |
Sternebra(e)- Fused |
0.9 |
0.78 |
0.9 |
0.0 |
3.1 |
0.0 |
2.6 |
40 |
35 |
Sternebra(e)- Malaligned1 |
7.1 |
3.11 |
7.6 |
2.9 |
10.2 |
@ |
@ |
48 |
31 |
Sternebra(e)- Malaligned (Slight or Moderate)2 |
5.5 |
5.19 |
2.9 |
0.0 |
16.6 |
0.0 |
16.3 |
207 |
125 |
Sternebra(e)- Malaligned (Severe)2 |
0.1 |
0.21 |
0.0 |
0.0 |
0.7 |
0.0 |
0.7 |
4 |
4 |
Sternum- Supernumerary Ossification Site |
0.3 |
0.47 |
0.0 |
0.0 |
1.5 |
0.0 |
1.4 |
13 |
12 |
Tarsal(s)- Unossified |
0.7 |
0.61 |
0.7 |
0.0 |
2.1 |
0.0 |
1.9 |
42 |
31 |
Vertebra- Supernumerary Site |
0.1 |
0.26 |
0.0 |
0.0 |
0.8 |
0.0 |
0.8 |
5 |
5 |
Vertebral Centra- Reduced Ossification |
0.4 |
0.62 |
0.0 |
0.0 |
2.4 |
0.0 |
2.1 |
14 |
12 |
1 Based on 4 datasets.
2 Based on 22 datasets.
@ Insufficient number of data for calculation.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- LOAEL
- 4.3 mg/kg bw/day
- Species:
- rat
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 439 mg/kg bw/day
- Species:
- rat
- Quality of whole database:
- Adequate for assessment.
Additional information
Information is available from six studies that have examined the developmental toxicity potential of Phenyl ethyl alcohol.
In an early investigation (Mankes et al., 1983), pregnant female Long-Evans rats received Phenyl ethyl alcohol at dose levels of 0, 4.3, 43, 432 mg/kg/day by oral gavage from day 6 to day 15 of gestation and the fetuses assessed on day 20. The highest dosage level caused maternal toxicity (intoxication), however the intermediate and low dose groups were asymptomatic. The incidence of malformed live fetuses was increased in a dose-related manner (50%, 93% and 100% of live offspring in the low-to-high dose groups), with pup weight and crown-rump length decreased reduced at 43 mg/kg bw/d and above. Mean live litter size was decreased in the low and intermediate dose but not in the high dose group. The NOAEL for maternal toxicity (intoxication) was 43 mg/kg bw/d, and the LOAEL for fetal effects (malformations, reduced live litter size) was 4.3 mg/kg/bw/day.
In an early pre-natal developmental toxicity study using the dermal route, Phenyl ethyl alcohol was applied to the skin of pregnant rats at dose levels of 0, 0.14, 0.43 and 1.40 ml/kg bw/d (equivalent to 0, 143, 439, 1428 mg/kg bw/d, based on density = 1.0202 g/cm3) during days 6-15 of pregnancy (Huntingdon Research Centre Ltd, 1986). The developmental status of the fetuses was assessed on day 20 of pregnancy. The results showed maternal and foetal toxicity at the highest dose tested. At 0.43 ml/kg bw/d, there were no clear effects on the mothers however foetal examination indicated a greater prevalence of skeletal variations and delays in ossification in treated animals versus controls. A treatment level of 0.14 ml/kg bw/d was associated with only equivocal evidence of effects on foetal skeletal development however, when viewed in the context of the study as a whole, the report concluded that the findings appeared part of a continuum of effects present in the intermediate and high dose groups. The NOAEL for maternal toxicity (mortality, decreased food consumption, reduced body weight) was 0.43 ml/kg bw/d (439 mg/kg bw/d), and the LOAEL for fetal effects (equivocal evidence of increased skeletal variations and delayed skeletal ossification) was 0.14 ml/kg bw/d (143 mg/kg/bw/day).
In another early pre-natal developmental toxciicty study via the dermal route (Argus Research Laboratories, Inc, 1988), Phenyl ethyl alcohol was applied to the skin of pregnant rats at treatment levels of 0, 0.07, 0.14, 0.28, 0.43 and 0.70 ml/kg bw/d (equivalent to 0, 71, 143, 286, 439 and 714 mg/kg bw/d, based on density = 1.0202 g/cm3) on days 6-15 of pregnancy, and foetal and litter parameters assessed on day 20. The NOAEL for maternal toxicity (excluding local irritation at the treatment site) and effects on litter parameters was the highest dose tested. Fetal findings were limited to significantly decreased body weights and delayed ossification of some skeletal components following treatment at doses of 0.14 ml/kg bw/d and above (although the extent of the effects seen at this dose level were quite minor). The NOAEL for maternal toxicity was 0.70 ml/kg bw/d (714 mg/kg bw/d), and the NOAEL for fetal effects (equivocal evidence of decreased fetal weights and delayed ossification of some skeletal components) was 0.07 ml/kg bw/d (71 mg/kg/bw/day).
In a combined dermal developmental and perinatal/postnatal reproduction toxicity study in the rat, Phenyl ethyl alcohol was applied to the skin of two series of pregnant rats at treatment levels of 0, 0.14, 0.43 and 1.40 ml/kg bw/d (equivalent to 0, 143, 439 and 1428 mg/kg bw/d, based on density = 1.0202 g/cm3, commencing on Day 7 of pregnancy and continuing until Day 20 (Charles River Laboratories, 2010). Fetal and litter parameters were assessed in one series of animals on Day 21 while mothers from the second series were allowed to deliver their litters normally. The dose level of 1.40 ml/kg bw was associated with clear maternal toxicity (e.g. mortality, decreased food consumption / body weight) and treatment of these groups therefore ceased on Day 15 of pregnancy. Litters from mothers administered PEA at 0.43 and 1.40 ml/kg bw and sacrificed on Day 21 of pregnancy showed decreased fetal skeletal ossification and an increased incidence of cervical ribs. In contrast, young rats from litters that had been delivered normally showed no evidence of these skeletal effects 3 weeks after birth (i.e. the changes were fully reversible). Based on these observations, 0.43 ml/kg bw/d (439 mg/kg bw/d, the highest dose tested throughout the whole study) was the NOAEL for maternal and fetal toxicity since any effects present in the offspring at birth were fully reversible.
In a supporting dose range finding pre-natal developmental toxicity study in time-mated female New Zealand White rabbits, Phenyl ethyl alcohol (in a 0.5% aqueous carboxymethyl cellulose + 1.25% Tween 80 vehicle) was adminitsered via oral gavage at dose levels of 0, 4.3, 43, 200 and 300 mg/Kg/day once daily, 7 days a week, from Day 6 to Day 28 post-coitum, inclusive. Three out of six females treated at 300 mg/Kg were sacrificed in extremis on Days 11-12 post-coitum, as they had no food consumption from the start of treatment resulting in a marked body weight loss (16-18%) along with absence of faeces production, because of the severe toxic effects all females were terminated early on Day 12 post-coitum due to animal welfare reasons. At 200 mg/Kg, food consumption was almost absent over the first days of treatment (Days 6-9 post-coitum), followed by a marked reduction in food consumption, especially up to Day 12 post-coitum (relative differences of 64% when compared to controls). This resulted in a significant body weight loss (average of 6%) over Days 6-9 post-coitum, followed by a statistically significantly reduced body weight gain over the entire treatment period and mean body weights were up to 10% lower when compared to controls. Therefeore, based on the results observed in this dose range finding prenatal developmental toxicity study, the maternal NOAEL for Phenyl Ethyl Alcohol was determined to be 43 mg/Kg.
A definitive pre-natal developmental toxicity study in time-mated female New Zealand White rabbits, where Phenyl ethyl alcohol (in a 0.5% aqueous carboxymethyl cellulose + 1.25% Tween 80 vehicle) was administered via oral gavage at dose levels of 0, 15, 50, 150 mg/Kg/day once daily, 7 days a week, from Day 6 to Day 28 post-coitum, inclusive, had reduced faeces production (up to severe) observed in animals across all the groups, including controls, with a higher persistence/severity at 150 mg/Kg/day. Treatment at 150 mg/Kg/day resulted in a mean body weight loss of 3% over Days 6-9 post-coitum, this decrease in body weight gain resulted in up to 6% lower mean body weights vs control throughout the study period, therefore, as complete recovery was not observed and mean fetal body weights were also reduced at this high dose level, the decrease in maternal body weight gain at 150 mg/Kg/day was considered to be adverse. At 150 mg/Kg/day, mean food consumption was significantly reduced (up to 53%) when compared to concurrent control mean during the first week of treatment, followed by a recovery from Day 15 post-coitum onwards. At 150 mg/Kg/day, mean combined fetal body weight was 10% lower vs control and mean values (in both male and females), this decrease in fetal body weights was observed at the same dose level at which lower maternal body weight gain from start of treatment until the end of pregnancy was also noted. The reduced fetal body weight was considered to be adverse due to the magnitude of the change indicative of intrauterine/foetal growth retardation. At 150 mg/Kg/day, an increase in the litter incidence of vertebral anomaly was observed in four fetuses and there was also an increase in the litter incidence of several skeletal variations as well as an increase in the litter incidence of the skeletal variation 13th full ribs and caudal shift of pelvic girdle. In conclusion, based on the results in this study,a maternal and developmental NOAEL for Phenyl Ethyl Alcohol was established at 50mg/Kg/day (based on the effects on maternal body weight gain and food intake, foetal body weight and skeletal malformation observed at 150 mg/Kg/day).
Justification for classification or non-classification
Additional information
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