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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:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed in line with good scientific principles, and in line with OECD 414 requirements.

Data source

Reference
Reference Type:
publication
Title:
Teratologic evaluation of 2-phenoxyethanol in New Zealand white rabbits following dermal exposure.
Author:
Scortichini BH, Quast JF & Rao KS
Year:
1987
Bibliographic source:
Fundamental and Applied Toxicology 8 (2): 272–279.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
The rabbit was selected as the more susceptible species
Principles of method if other than guideline:
New Zealand White rabbits (25 per group) were dosed dermally (occlusive) with 2-phenoxyethanol at concentrations of 300, 600 or 1000 mg/kg bw/day from GD 6 (after artificial insemination) to gestation day 18. Does were examined for signs of toxicity and body weights, urinalysis and haematological parameters were assessed as part of the study. At termination on gestation day 28, foetuses were removed by caesarean section and examined for abnormalities. The uterine horn was removed and examined. Maternal livers were removed and weighed.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Reference substance name:
2-phenoxyethanol
EC Number:
204-589-7
EC Name:
2-phenoxyethanol
Cas Number:
122-99-6
IUPAC Name:
2-phenoxyethanol
Test material form:
other: liquid (not specified)
Details on test material:
- Name of test material (as cited in study report): 2-phenoxyethanol
- Physical state: Liquid
- Analytical purity: > 99 %
- Specific gravity: 1.1

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 3.5-4.5 kg
- Housing: Singly in wire bottomed cages
- Diet: ad libitum
- Water: municipal tap water ad libitum
- Acclimation period: At least two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 22 °C
- Humidity (%): relative humidity 50 %
- Photoperiod (hrs dark / hrs light): 12 hour light/dark photocycle

Administration / exposure

Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: Back
- Type of wrap if used: Occlusive. The wrap consisted of an absorbent piece of gauze and non-absorbent piece of cotton covered with a cotton flannel bandage held in place with tape.
- Time intervals for shavings or clippings: Initially the animals were clipped prior to inseminitation. Prior to each application the area was examined for irritation or re-growth of hair. The application site was clipped as needed throughout the study.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The test material was renewed daily. At the end of the exposure, the bandages were removed and the application site was washed with water to remove any residual test material and to prevent oral ingestion.
- Time after start of exposure: Gestation day 19

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.27, 0.55 and 0.91 mL/kg for 300, 600 and 1000 mg/kg bw/day respectively.

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: artificial insemination (noted as gestation day 0)
Duration of treatment / exposure:
Gestation days 6 to 18
Frequency of treatment:
Daily
Duration of test:
Up to gestation day 28
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (distilled water control), 300, 600 and 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
25 rabbits (does) per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were selected on the results of a previous study where groups of 10 animals were treated with 0, 300, 600 or 1000 mg/kg bw/day. Minimal maternal toxicity was observed (lower bodyweight) at 1000 mg/kg bw/day. The top dose was selected due to the physical constraints of dosing a higher dose level dermally.

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal bodyweights were recorded on gestation day 6 to 19 and gestation day 28 (termination.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from an ear vein on gestation day 19 and when killed in extremis.
- How many animals: approximately 10 per group on day 19 of gestations and 3 animals (2 at 600 and 1 at 1000 mg/kg bw/day) sacrificed in extremis.
- Parameters: Packed cell volume (PCV), haemoglobin (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.

URINALYSIS: Yes
- Time schedule for collection of urine: At necropsy from the bladders of two moribund rabbits (one from each of the 600 and 1000 mg/kg bw/day groups).
- Metabolism cages used for collection of urine: No, urine was collected from the bladders via aspiration.
- Parameters: Colour, appearance, specific gravity, pH, protein, glucose, ketones, billirubin, blood, urobilinogen, white blood cells, red blood cells and microscopic examination for crystals and epithelial cells.
Ovaries and uterine content:
The uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Uteri were also examined for live or dead foetuses.
Fetal examinations:
- External examinations: Yes: all foetuses were weighed, measured (crown-rump length), sexed and examined for external alterations.
- Soft tissue examinations: Yes: One half of each litter (selected randomly) were examined under a dissecting stereomicroscope for visceral alterations.
- Skeletal examinations: Yes: all foetuses were stained with alizarin red S and examined for skeletal alterations.
Statistics:
Analyses of maternal and foetal bodyweights, absolute and relative organ weights, applicable haematological parameters, and foetal length were performed using a parametric or non-parametric analysis of variance followed by either a Dunnett’s test or the Wilcoxon rank sum test with Bonferroni’s correction. The frequency of pre-implantation loss, resorptions among litters and foetal population and foetal alterations was assessed using a censored Wilcoxon test with Bonferroni’s correction. Implants, litter size and corpora lutea were analysed with a nonparametric analysis of variance followed by the Wilcoxon rank sum test with Bonferroni’s correction. The Fisher’s exact probability test was used to analyse pregnancy rates. Foetal sex ratios were assessed by a binomial distribution test. Statistical significance was considered to be 0.05.

As numerous measurements were statistically compared in the same group of animals, the overall false positive rate was reported to be greater than the level of significance. Therefore the results of the study were interpreted based on the statistical analyses of the numerical data and also considering other factors such as dose-response relationships and the significance of the observations when compared to other findings.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: A number of rabbits in the 600 and 1000 mg/kg bw/day died or were moribund. At necropsy the animals were observed to have intravascular haemolysis.

Details on maternal toxic effects:
- Mortality: Five rabbits in the 600 mg/kg bw/day group and nine in the 1000 mg/kg bw/day group died or were killed in extremis. The majority of deaths occurred in the period between gestation day 11 and 18 (6 to 13 doses). The salient pathologic finding in most of these animals was dark urine in the bladder. In addition, the animals were jaundiced and the kidneys were darkened in colour. The gross necropsy findings were typically observed to be associated with an intravascular haemolytic episode. In the moribund animals which were assessed for haematological parameters, RBC counts and PCV values were severely depressed whereas reticulocytes were elevated. In addition, red blood cell fragility was increased. The observed pathological changes were interpreted as regenerative haemolytic anaemia. The dark urine at gross necropsy was concluded to be haemoglobinuria as there were no intact RBCs in the urine sediment. No other significant haematologic findings were observed except for those related to intravascular haemolysis. The specific cause of death for two animals in the 600 mg/kg bw/day group and one in the 1000 mg/kg bw/day group could not be determined at necropsy. There were no indications of intravascular haemolysis. The 1000 mg/kg bw/day group was terminated due to the excessive lethality observed.
- Local irritation: Throughout dosing, slight to moderate reddening of the skin at the application site was seen in some animals in all groups. Four does in the 600 mg/kg bw/day group and three in the 1000 mg/kg bw/day group had darkened areas of skin at the application site. Staining in the perineal region and/or the presence of dark coloured urine underneath the cages was noted for several animals in the 600 and 1000 mg/kg bw/day group.
- Necropsy: Treatment was staggered; five animals therefore completed the dosing regimen in the 1000 mg/kg bw/day group before the group was terminated. In these animals, no adverse effects were observed in the foetuses (no statistical analyses were performed) or in the maternal animals.
- Haematology: To further assess the haemolytic changes at 600 and 1000 mg/kg bw/day, blood for haematological examinations was drawn from the remaining animals at the end of treatment. None of the animals exhibited the same intravascular haemolysis effects.
- Bodyweights: The pregnant rabbits in the 300 mg/kg bw/day group weighed significantly more than the controls prior to the study initiation. This continued throughout the study. No differences in bodyweight gains, absolute liver weights and relative livers weights were noted between the treated and the control rabbits.
- Reproductive parameters: No adverse effects on implantations resorbed or foetal measurements were observed at 300 or 600 mg/kg bw/day.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: No effects were noted at 600 mg/kg bw/day (a maternally toxic dose).

Details on embryotoxic / teratogenic effects:
Examination of external, visceral or skeletal malformations did not indicate a teratogenic effect of the test material up to 600 mg/kg bw/day. Single foetuses from the control group exhibited microphthalmia and anonychia. No organ malformations were noted in any of the animals in both the control and treated groups. Skeletal alterations in the treated group occurred at the same frequency as the control group. One foetus in the control group exhibited oligodactyly and one foetus in each of the two treated groups had clinodactyly. Hemivertebra was noted in one foetus at 600 mg/kg bw/day.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Haematological parameters

2-phenoxyethanol (mg/kg bw/day)

0

300

600

Haematologic parameters measured on GD 19

N

10

10

10

RBC (x 106/mm3)

5.97 ± 0.29

5.79 ± 0.29

5.94 ± 0.54

Hgb (g/dL)

13.1 ± 0.8

12.9 ± 0.5

13.4 ± 0.9

PCV (%)

46.4 ± 2.6

44.8 ± 1.9

46.1 ± 3.1

Platelets (x 103/mm3)

454 ± 93

422 ± 83

393 ± 109

Reticulocytes (%)

3.2 ± 1.1

2.3 ± 0.8

3.0 ± 1.3

WBC (x 103/mm3)

7.0 ± 1.5

7.2 ± 1.7

6.3 ± 1.7

2-phenoxyethanol (mg/kg bw/day)

600

600

100

Haematologic parameters measured in moribund animals

No. doses received

8

6

6

RBC (x 106/mm3)

0.59

0.63

0.97

Hgb (g/dL)

3.2

3.3

3.2

PCV (%)

6.3

6

10.6

Platelets (x 103/mm3)

465

1266

785

Reticulocytes (%)

21.5

19.4

-

WBC (x 103/mm3)

10.4

21.5

9.4

 

Table 2: Maternal toxicity and reproductive parameters

2-phenoxyethanol (mg/kg bw/day)

0

300

600

1000a

No. deaths/No. females

0/25

0/25

5/25

-

Pregnancies detected by stain

1

0

1

-

% Pregnant

76

80

88

-

No. of litters

18

20

16

5

Corpora lutea/dam

11 ± 3

11 ± 2

11 ± 2

11 ± 3

Implantations/dam

9 ± 3

8 ± 3

10 ± 3

11 ± 2

Live foetuses/litter

7 ± 4

7 ± 3

9 ± 3

10 ± 3

% Implantations resorbed

22 (36/164)

12 (19/161)

11 (17/153)

9 (5/54)

Foetal bodyweight (g)b

34.2 ± 4.7

36.6 ± 7.1

32.5 ± 6.8

33.0 ± 4.8

Foetal crown rump lengthb

84.3 ± 4.2

86.8 ± 5.9

84.0 ± 5.8

84.5 ± 1.4

Maternal bodyweight GD6 (g)

3827 ± 244

4079 ± 286*

4018 ± 245

4047 ± 301

Maternal bodyweight gain (g)

GD

6-9

-54 ± 115

-55 ± 84

-61 ± 104

-51 ± 101

9-12

51 ± 93

29 ± 83

6 ± 95

-8 ± 82

12-15

13 ± 84

-16 ± 234

29 ± 90

41 ± 63

15-19

-32 ± 84

49 ± 217

-71 ± 164

41 ± 118

19-28

38 ± 215

74 ± 151

118 ± 169

73 ± 269

6-19

-22 ± 240

8 ± 137

-97 ± 174

22 ± 222

6-28 (Total)

16 ± 368

86 ± 192

21 ± 253

95 ± 81

Absolute maternal liver weight (g)

107.03 ± 23.05

110.98 ± 21.07

106.10 ± 21.03

106.11 ± 11.20

Relative maternal liver weight (g)

2.78 ± 0.53c

2.67 ± 0.47

2.63 ± 0.41

2.57 ± 0.30

aNot used in statistical evaluations

bAssessed per litter

cmean ± SD (g organ wt/100 g body wt)

* Significantly different from control value ( = 0.05)

 

Table 3: Foetal (teratogenicity) observations

2-phenoxyethanol (mg/kg bw/day)

0

300

600

1000a

No. foetuses (No. litters) examined

External and skeletal examinations

128 (17)

142 (20)

136 (15)

49 (5)

Visceral examinations

72 (17)

80 (20)

70 (15)

26 (5)

No. foetuses (No. litters) affected

Anonychiab

1 (1)

0

0

0

Microphthalmiab

1 (1)

0

0

0

Forelimb flexure, mild

0

0

1 (1)

1 (1)

Dilated renal pelvis

0

1 (1)c

2 (2)

0

Retrocaval ureter

2 (2)

5 (3)

4 (2)

0

Pale streaks in kidney

0

1 (1)c

0

0

Accessory adrenal

1 (1)

0

0

0

Small Spleen

0

0

1 (1)d

0

Pale spleen

0

0

1 (1)d

0

Skull

Delayed ossification

4 (3)

1 (1)

5 (2)

1 (1)

Foramen

5 (2)

3 (3)

1 (1)

2 (1)

Hyoid, delayed ossification

72 (15)

61 (17)

76 (15)

24 (4)

Hyoid, crooked

12 (9)

16 (8)

17 (11)

1 (1)

Vertebrae

Delayed ossification

0

2 (2)

1 (1)

0

Hemivertebra

0

0

1 (1)

0

Cervical spur

0

2 (1)

0

0

Lumbar spur

25 (12)

43 (17)

27 (9)

8 (5)

Axis, dentoid process, delayed ossification

6 (3)

8 (4)

10 (6)

3 (1)

Centra

Delayed ossification

1 (1)

2 (2)

2 (1)

0

Extra site of ossification

0

1 (1)

0

0

Stenebrae

Delayed ossification

54 (13)

57 (18)

67 (15)

18 (5)

Fused

1 (1)

2 (1)

2 (2)

0

Irregular pattern of ossification

0

0

1 (1)

0

Asymmetric cartilage

0

1 (1)

0

0

Extra band of cartilage

1 (1)

0

0

0

Other

Thumb digit delayed ossification

1 (1)

0

0

0

Oligodactylb

1 (1)

0

0

0

Clinodactylb

0

1 (1)

1 (1)

0

Pelvic bones, delayed ossification

0

0

4 (1)

0

aNot used in statistical evaluations

bConsidered to be a malformation

cOne foetus had pale streaks in the kidney and a dilated renal pelvis

dOne foetus had a small pale spleen

 

Applicant's summary and conclusion

Conclusions:
Dermal application of 2-phenoxyethanol in pregnant rabbits did not produce any evidence of teratogenicity, foetotoxicity or embryotoxicity at 600 mg/kg bw/day, a dose which was observed to be maternally toxic. No adverse maternal or foetal effects were noted at 300 mg/kg bw/day. Under the conditions of the test, the NOAEL for developmental toxicity and teratogenicity was determined to be 600 mg/kg bw/day. The NOAEL for maternal toxicity was determined to be 300 mg/kg bw/day.
Executive summary:

New Zealand White rabbits (25 per group) were dosed dermally with 2-phenoxyethanol at concentrations of 300, 600 or 1000 mg/kg bw/day from gestation day 6 (after artificial insemination) to gestation day 18. The test material was renewed every 24 hours and covered with an occlusive wrap. Does were examined for signs of toxicity and body weights, urinalysis and haematological parameters were assessed as part of the study. At termination on gestation day 28, foetuses were removed by caesarean section and examined for abnormalities. At this time, the uterine horn was removed and examined. Maternal livers were removed and weighed.

Nine of the animals in the 1000 mg/kg group were found dead or killed in extremis; this group was terminated early in the interests of welfare but a total of 5 does was still available for caesarean sectioning at gestation day 28. Five animals in the 600 mg/kg group also were found dead or killed in extremis. Maternal toxicity was observed at 1000 mg/kg, in the form of intravascular haemolysis. Maternal toxicity was noted in the 600 mg/kg group, but not at the same severity. Rabbits in the two highest dose groups surviving to the end of the study had no treatment related effects. The test substance was found not to be embryotoxic, foetotoxic or teratogenic.