Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was comparable to a guideline study with acceptable restrictions.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
substance was applied dermally and the uterine weight was not recorded.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): 2-phenoxyethanol
- Analytical purity: >99%

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Animals were allowed to acclimitise to laboratory conditions for 2 weeks prior to breeding. The species was chosen as it has been shown to be more sensitive to ethylene glycol monomethyl ether than both rats and mice. Animals were singly housed in wire bottom cages and temperature was maintaned at 22 degrees C and relative humidity 50% with 12 hr dark/light photioperiod. Female rabbits were approxiomately 3.5 to 4.5 kg at study intiation. Feed and water was available ad libitum throughtout the study. Animals were randomly assigned into groups using a computer generated system. Animals were uniquely identified using metal ear tags.

Administration / exposure

Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
Prior to insemination on day 0 of gestation, a section on the back of each rabbit was clipped with electric clippers. The test material was applied to to the clipped area daily beginning day 6. The application site was occluded using a piece of absorbent gauze and nonabsorbent cotton covered by a cotton flannel bandage held in place with tape. The bandages remianed in place 24 hours a day during the treartment period. Prior to daily application the skin was assessed for signs of irritation and hair regrowth. Bandages were replaced and hair reclipped as needed during the treatment period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis by gas chromatography was conducted on the test material, it was found to have a purity of > 99%. Test item was applied neat.
Details on mating procedure:
Impregnation procedure: Test animals were artificially inseminated.
Duration of treatment / exposure:
once daily from day 6 through to day 18 of gestation.
Frequency of treatment:
once daily from day 6 through to day 18 of gestation.
Duration of test:
28 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
other: dose volume of test item applied 0.27 ml/kg (2-phenoxyethanol specific gravity = 1.1)
Remarks:
Doses / Concentrations:
600 mg/kg/day
Basis:
other: dose volume of test item applied 0.55 ml/kg (2-phenoxyethanol specific gravity = 1.1)
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
other: dose volume of test item applied 0.91 ml/kg (2-phenoxyethanol specific gravity = 1.1)
No. of animals per sex per dose:
25 animals/dose group
Control animals:
other: distilled water (0.91 ml/kg BW)
Details on study design:
Control animals were treated with distilled water at a targeted volume of 0.91 ml/kg body weight.

The dose volume of undiluted 2-phenoxyethanol (specific gravity = 1.1) was 0.27, 0.55 and 0.91 ml/kg for the 300, 600 and 1000 mg/kg/day dose levels. The selection of dose levels was based on a results of a previous study in which groups of 10 animals were treated with 0, 300, 600 and 1000 mg/kg/day of phenoxyethanol on days 6 through to 18 of gestation. Minimal maternal toxicity was evidenced by a lower bodyweight gain through days 15 to 18 of gestation at 1000 mg/kg/day. The highest dose was selected as 1000 mg/kg/day due to physical constraints (application of a liquid to a finite area of the animals back without excessive run off at the time of bandage application or subsequent run off from the occluding bandage.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: On gestation day 6 through to 19 and on day 28.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #28
- Organs examined:

Blood was collected from the ear vein from approximately 10 animals per dose group on day 19 of gestation and 3 animals (2 at 600 and 1 at 1000 mg/kg/day) were scarificed in extremis for the follwoing measurements: packed cell volume (PCV), hemaglobin (Hgb), erythrocyte count (RBC), total leukocyte count (WBC), red blood cell indices (MCV, MCH, MCHC), platelet count (PLAT), reticulocyte count, osmotic red cell fragility and WBC differential counts. Urine was collected at the time of necropsy from the bladders of two moribund rabbits (one each at 600 mg/kg/day and 1000 mg/kg/day via aspriation for urinalysis (colour, appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, white blood cell, red blood cells and microscopic examination for crystals and epithelial cells). Maternal liver weights were recorded at the time of cesarean section on day 28 of gestation.
Ovaries and uterine content:
Following caesarean section, the number of corpora lutea and the number and position of implantations, resorptions and live or dead fetuses were recorded. The uteri of apparently non pregnant females were stained with a 10% solution of sodium sulphide and examined for evidence of early implantation sites. This procedure was carried solely to determine the indicies of pregancy and not to evaluate indices of reabsorption.
Fetal examinations:
All fetuses were weighed, measured (crown rump length), sexed and examined for external alterations. One half of each litter, selected using a table of random numbers were examined under dissecting stereomicroscope for evidence of visceral alterations. All fetuses were then preserved in 95% ethanol, cleared, stained with alizarin red S and examined for skeletal alterations.
Statistics:
Statistical evaluation: Analysis of maternal and fetal body weights, absolute and relative organ weights, appropriate hematologic parameters and fetal length were performed using a parametric or nonparametric analysis of variance followed by a Dunnett's test or the Wilcoxon rank sum test with Bonferroni's correction where appropriate. Evaluation of preimplantation loss, resorptions, and fetal alterations were analyzed by a censored Wilcoxon test with Bonferroni's correction. Corpora lutea, implants and litter size were analyzed with a nonparametric analysis of variance followed by the Wilcoxon rank sum test with Bonferroni's correction. Pregnancy rates were analyzed by the Fisher's exact probability test and fetal sex ratios were analyzed by a binomial distribution test. The nominal value used for statistical evaluation was p<0.05.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Throughout the dosing period slight to moderate reddening of the skin at the application site was seen in some animals at all treatment levels. Four rabbits in the 600 mg/kg/day dose group and three in the 1000 mg/kg/day were observed to have darkened area of skin at the application site. Staining in the peritoneal region and/or presence of dark coloured urine underneath their cages was noted in several animals in the middle and high dose groups.

During the study five rabbits in the 600 mg/kg/day and 9 rabbits in the 1000 mg/kg/day dose groups died or were sacrificed in extremis. Most deaths occured between gestation days 11 and 18 (6 to 13 doses). Pathological findings in most of these animals were dark coulred urine in the bladder, jaundice and darkened kidneys. The gross necropsy findings were those typically observed to be associated with an intravascular hemolytic episode. In those moribund animals were hematological parameters were investigated , RBC counts and PCV values were severly depressed, whereas reticultocytes were elevated. In addition red blood cell fragility was increased. The collective data indicated the presence of a regenerative hemolytic anemia in these rabbits. The dark urine observed at gross necropsy was interpretated to be due to hemoglobinuria as there was no intact red blood cells in the urine sediment. The specific cause of death for three animals. two in the 600 mg/kg/day and 1 in the 1000 mg/kg/day could not be determined under gross necropsy. Due to the excessive lethality seen in the 1000 mg/kg/day group the remaining animals were terminated for humane reasons with no further observations. Due to the staggered nature of the initiation of treatment five rabbits at 1000 mg/kg/day had been sacrificed on day 28 of gestation, prior to the decision to terminate. Fetuses from these five rabbits were examined for external, visceral and skeletal alterations. No statistical evaluation was performed on this data however no evidence of adverse effect was found in these five rabbits or their concepti.

To further assess thr hemolytic changes observed in the 600 mg/kg/day and 1000 mg/kg/day dose levels, blood from hematological evalaution was drawn from the remaining animals on study upon completion of treatment period (day 19 of gestation). None of the parameters measured was adversely affected in surviving animals.

Pregnant rabbits in the 300 mg/kg/day group weighed signficantly more than the controls prior to the start of the study, a similar trend continued throughout most of the experiemntal period. No differences in body weight gains or absolute or relative liver weights were seen between controls and treated rabbits.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
600 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
> 600 mg/kg bw/day
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No adverse effects were seen on pregnancy rates, implantation resorbed or fetal body measurements were observed among rabbits dosed dermally with 300 or 600 mg/kg/day. Similarly the indices of malformations observed externally, viscerally or at skeletal examination gave no indication of a teratogenic response to 2-phenoxyethanol at levels up to 600 mg/kg/day. In the control group single fetuses from different litters exhibited microphthalmia and anonchyia. No malformations of the internal organs were seen in control or treated groups. Alteratations of the skeletal system occured with similar frequency in the control and treated groups. One control fetus exhibited oligodactyly (missing digit) and one fetus in each of the two treated groups had clinodactyly (lateral deflection of digit). One fetus in the 600 mg/kg/day dose group had a hemivertebrae.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Dermal application of 2-phenoxyethanol produced no evidence of teratogenicty, fetotoxicity or embryotoxicity at 600 mg/kg/day, a dose that was observed to be maternally toxic.
Executive summary:

Maternal toxicity as evidenced by intravascular hemolysis of red blood cells and death was seen in pregnant rabbits exposed dermally to 600 and 1000 mg/kg 2-phenoxyethanol/kg/day. Maternal toxicity occured in dose related manner with nine dead or moribund animals in the 1000 mg/kg/day group and five at 600 mg/kg/day, no deaths occured in the 300 mg/kg/day group. Subsequent to the onset of clinical signs, death followed rapidly, usually within a 24 hour period. Intravascular hemolysis was diagnosed in these animals based on observed changes in hematology and urinalysis parameters and the associated gross pathology. Rabbits in the 600 and 1000 mg/kg/day dose groups which survived to day 28 of gestation showed no effects. No signs of adverse maternal effects were seen at 300 mg/kg/day.

Single occurences of hemivertebra and clinodactyly were observed among the litters of the dosed rabbits. The low incidences of those malformations were considered to be sporadic occurences and not indicative of a treatment related effect. In conclusion, the dermal application of 2-phenoxyethanol produced no evidence of teratogenicity, fetotoxicity or embryotoxicity at 600 mg/kg/day, a dose that was observed to be maternally toxic. In addition no adverse effects on teratogenicity, fetotoxicity or embryotoxicity were seen at 1000 mg/kg/day, however too few animals survived to render these result statistically significant, therefore the dose of 600 mg/kg/day was selected as the NOAEL.

The data presented for material 2 -phenoxyethanol (EC 204-589-7) is considered appropriate for read-across to EC 202-228-8 and for use in the generation of DNELs and concluding on classification. The justification for the use of this read across is located within IUCLID section 13.