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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
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: see 'Remark'
Remarks:
The study was performed in line with GLP and the method was designed in line with good scientific principles. The study predates the inception of the OECD guideline 416 (2-generation reproductive toxicity), but is in basic compliance with the current standardised guideline. There is a good level of detail in reporting, sufficient to assess the quality of the data.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
The reproductive toxicity of the test substance was investigated in Swiss CD-1 mice. Mice were exposed to 2-phenoxyethanol in diet at concentrations of 0, 0.25, 1.25 and 2.5% in diet equivalent to 0, 0.4, 2.0 and 4 g/kg bw/day (a range finding study was conducted with concentrations of 0, 1, 2.5, 5, 7.5 and 10% in feed for 2 weeks). Observations during the study included general toxicity, sperm analysis, reproductive performance, pup abnormalities and assessment of possible sex related effects (crossover mating).
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P) 11 wks at the beginning of the continuous breeding test; (F1) 74 ± 10 days.
- Housing: Animals were housed by sex in solid bottom polypropylene or polycarbonate cages with stainless steel lids during the pre-mating period. The animals were then housed either individually or in breeding pairs.
- Diet (e.g. ad libitum): ad libitum.
- Water: Deionized water available ad libitum.
- Acclimation period: 2 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C.
- Photoperiod (hrs dark / hrs light): 14 hours light (10 hours dark).
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: Weekly (diet was shown to lose approximately 5 % over 7 days).
- Mixing appropriate amounts with (type of food): Each dose level was blended into a small amount of ground diet, which was then added to a pre-weighed portion and mixed.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 98 days as breeding pairs in the continuous breeding phase, in the crossover trial and the offspring fertility assessment animals were cohabited for 7 days
- Proof of pregnancy: vaginal plug
- After successful mating each pregnant female was caged (how): In the offspring fertility test, animals were housed individually after the cohabitation period. During the continuous breeding phase, the animals cohabited for 98 days.
- Any other deviations from standard protocol: In the crossover mating trial, the parental animals from the continuous breeding phase were cross mated with control animals to determine the affected sex. Three groups of 20 pairs were used: control males x control female; high dose males x control females; control males x high dose females. In the offspring fertility check, the last litter produced from the continuous breeding phase and cohabited with a mate from a different litter within the same dose group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No details of the methods used were reported. Aliquots of six representative samples were analysed during the study, the concentration was shown to be within 96 - 105% of the nominal concentration.
Duration of treatment / exposure:
- Range finding test: 14 days.
- Continuous breeding study: 7 day pre-mating dosing followed by 98 days cohabitation.
- In the cross-over mating trial parental animals selected from the continuous breeding study were necropsied after 3 weeks.
- The animals selected for the offspring fertility test were necropsied at the end of the test (after delivery).
Frequency of treatment:
Daily, dosed feed was available ad libitum, apart from the 7 days were breeding pairs were cohabited in the cross-over mating trial.
Details on study schedule:
- F1 parental animals not mated until 74 ± 10 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
Remarks:
Doses / Concentrations:
4000, 2000, 400 and 0 mg/kg bw /day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 0.25, 1.25 and 2.5 %
Basis:
actual ingested
No. of animals per sex per dose:
- 8 animals/sex/group in each of the range finding dose levels
- 40 breeding pairs in the control group and 20 breeding pairs per dose were using in the continuous breeding phase of the study.
In the crossover mating test 20 breeding pairs per group (3 in total) were used.
- In the offspring fertility test, 8 to 10 litters from the F1 generation from the control group and the 1.25 % dose level were selected for rearing to sexual maturity. At 74 ± 10 days one to three female and male pups were selected from each surviving litter and selected randomly for breeding. 19 breeding pairs per dose were used in this test.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: A 14-day range finding study was included in order to select an appropriate dose for the study. Five dose groups and a control were exposed to 10, 7.5, 5.0, 2.5, 1.0 and 0.0 % in diet for two weeks. Animals were assessed for clinical signs, bodyweight changes and food consumption. Based on the results of this study, the appropriate doses were selected for the continuous breeding study. As both male and female animals lost 10 % of their bodyweight in the 7.5 and 10 % groups; 0, 0.25, 1.25 and 2.5 % were selected as the dosing levels in the continuous breeding trial.
Parental animals: Observations and examinations:
All animals (continuous breeding study, crossover mating trial and the offspring fertility test) were assessed for the following.

CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes calculated as the average g/day/mouse
Sperm parameters (parental animals):
The following parameters examined in all male mice in all tests within the study; sperm count, sperm motility and sperm morphology.

In addition, the following were examined in the continous breeding test and the offspring fertility test (F0 and F1) animals; testis weight and epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring; number and sex of pups, stillbirths, live births, postnatal mortality, weight gain and fertility.

The number of litters produced by the F0 generation in the continuous breeding phase of the study was also evaluated.

GROSS EXAMINATION OF DEAD PUPS: no
Postmortem examinations (parental animals):
ORGAN WEIGHTS
In the male animals from the continuous breeding study, the females from the crossover mating test and both the males and females from the offspring fertility test the liver weights were recorded.

In addition the following organs and tissues from male animals in the continuous breeding test and the offspring fertility test were weighed: right epididymis, right testis, seminal vesicles and prostate.
Postmortem examinations (offspring):
ORGAN WEIGTHS
As listed above, the following organ weights from the pups produced in the continuous breeding study used in the offspring fertility test were examined: liver, right epididymis, right testis, seminal vesicles and prostate.
Statistics:
The findings from the continuous breeding study were assessed using the Cochran-Armitage test to determine whether there was a dose-related trend. In the cross-over mating trial, as it was not possible to test for a dose-related trend, the findings were assessed using a Chi squared test for homogeneity to determine the overall difference in fertility among groups. Fisher’s exact test was As a pariwise comparisons between the control and dosed groups.
Dose groups means for sex ratio and proportion of live pups were assessed using the Kruskal-Wallis test to examine overall difference and Jonckheere’s test to assess ordered differences. Pairwise comparisons of treatment group means were performed using the Wilcoxon-Mann-Whitney U test.

To prevent the number of pups per litter affecting the assessment of average pup weight an analysis of covariance was used, using the average litter size and the covariate including live and dead pups. Least-squares estimated of dose group means, adjusted for litter size were tested using an F test to assess overall equality and a t test to test for pairwise equality. To prevent interference from potential sex differences, these were performed with male and females separately and also both sexes combined.

An analysis of covariance was also used to adjust organ weights appropriately for bodyweight. Unadjusted body and organ weights were analysed by the Kruskal-Walls and the Wilcoxon-Mann-Whitney U tests. Jonckheere’s test was used to test for dose-related trends.
Reproductive indices:
The following reproductive indices were calculated using equations below:

Mating index (%) = (Number of animals with copulatory plug/Number of animals cohabited breeding pairs) x 100

Fertility index (%) = (Number of fertile pairs/Number of animals with copulatory plug) x 100
Offspring viability indices:
The number of litters and live pups per litter were assessed per fertile breeding pair from this figure the treatment group means were determined. The proportion of live pups was defined as the number of pups born alive divided by the total number of pups produced by each breeding pair. The sex ratio was expressed as the proportion of male pups born alive out of the total number of live pups born to each fertile pair.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Three females died in the continuous breeding study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males only.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males only.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: No treatment related effects.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No treatment related effects.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
2.5 % EGPE in diet reduced the number of litters delivered per breeding pair and reduced litter size and number of live births.
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
The test substance was found to have a low general toxicity, with only three female dying in the continuous breeding test.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the continuous breeding study, only a minimal effect was noted on male bodyweight (2 % decrease) with no change on average in females. There was no significant effect on feed consumption. The approximate daily consumption was 5.6 g/day/mouse. At necropsy at the end of the cross-over mating test, there was a significant decrease in the bodyweight for males exposed to 2.5 % EGPE.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
There were no effects on feed consumption. The average daily test substance intake was approximately 400, 200 and 4000 mg/kg bw/day from the 0.25, 1.25 and 2.5 % groups respectively. The test substance intake varied in the females with the stage of gestation due to fluctuations in bodyweight.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
In the Cross-over mating test there were no differences between the treated and control males in respect to sperm concentration, percentage of motile sperm and percentage of abnormal sperm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Continuous exposure in the continuous breeding test did not affect the number of breeding pairs able to produce at least one litter. Exposure to 2.5 % of EGPE was found to reduce the number of litters delivered per breeding pair and significantly reduced the size and proportion of pups born alive. Further assessment of the litters demonstrated that in the high-dose group only 12 out of 20 pairs (60 %) had a fifth litter compared to 36 out of 40 (90 %) in the control. The number of pups per litter was not found to be affected. In the cross-over mating test, the results did not show which sex was affected by administration of the test material. The only difference was live pup weight was significantly decreased by 12 % in the control males crossed with the 2.5 % dosed females.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In the cross-over mating trial, when adjusted for bodyweight, the liver weight was found to be significantly increased in both males and females (21 and 60 % respectively). There were no significant difference in weight of the right testis, prostate and epididymis between controls and the 2.5 % EGPE males.
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight, liver weight and number of litters produced, live pup weight and litter size
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
High lethality was seen in the 2.5 % group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dose related decrease in body weight.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment related effects.
Gross pathological findings:
not examined
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
Exposure to 2.5 % of EGPE in the continuous breeding test was found to reduce the number of litters delivered per breeding pair and significantly reduced the size and proportion of pups born alive. Further assessment of the litters demonstrated that in the high-dose group only 12 out of 20 pairs (60 %) had a fifth litter compared to 36 out of 40 (90 %) in the control. The number of pups per litter was not found to be affected. In the final litters produced by the continuous breeding test, pup lethality was pronounced during the lactation and post-weaning periods in the 1.25 and 2.5 %. By day 21 (weaning) only eight litters in the 1.25 and 2.5 % dosing groups had sufficient pups for the offspring fertility test. From birth to the start of mating, 12 of 87 (14 %) of the selected offspring for the fertility test in the control group, 20 of 113 pups (18 %) in the 0.25 % EGPE group, 33 out of the 84 pups (39 %) in the 1.25 % EGPE dose group and 66 out of the 76 pups (87 %) in the 2.5 % EGPE treatment group had died. Due to the high lethality in the 2.5 % group (25 of 32 males and 21 of 24 females), the offspring fertility test was performed with the pups from the 1.25 % dosing group.

BODY WEIGHT (OFFSPRING)
There was a significant dose related decrease in adjusted live pup weight during the continuous breeding test. In the cross-over mating test live pup weight was significantly decreased by 12 % in the control males crossed with the 2.5 % dosed females. The F1 pups selected for the offspring fertility test (the last litters produced by the continuous breeding test) were found to have dose-related decreased bodyweights at birth, weaning and at mating (the start of the offspring fertility test). At the end of the offspring fertility test, bodyweights were found to be decreased in both males and females dosed with 1.25 % EGPE.

SEXUAL MATURATION (OFFSPRING)
In the offspring fertility test, there were no statistically significant effects on the proportion of copulatory plug positive matings (mating index), fertile pairs (fertility index), pups born alive, or number of pups per litter when compared to the control pairs. Consistently, the live pup weights for the F2 generation were reduced. The offspring fertility test did not indicate any effects on sperm concentration, percentage of motile sperm or percentage of abnormal sperm.

ORGAN WEIGHTS (OFFSPRING)
The F1 pups used in the offspring fertility test were found to have increased liver weights when adjusted for bodyweight at the end of the test. There was no effect on right testis, prostate or epididymal weight. Seminal vesicle weight was found to be significantly decreased compared to controls.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight, liver weight and live pup weight
Reproductive effects observed:
not specified

Table 1: Reproductive performance

Parameter

Continuous breeding test

Crossover mating test

Offspring fertility test

Control

0.25% EGPE in diet

1.25% EGPE in diet

2.5% EGPE in diet

Control male x Control female

2.5 % treated male x control female

Control male x 2.5 % treated female

Control

1.25% EGPE in diet

No. with copulatory plugs/No. cohabited

 

18/18

18/20

14/17

18/19

19/19

Mating index (%)

100

90

82

95

100

No. fertile/No. with copulatory plugs

38/38

20/20

19/20

18/18

16/18

16/18

12/14

17/18

18/19

Fertility index (%)

88

88

86

94

95

Litters per breeding paira

4.8 ± 0.1

4.8 ± 0.1

5.0 ± 0.1

4.5 ± 0.2

Live pups per littera

11.0 ± 0.4

11.6 ± 0.5

11.3 ± 0.5

9.0 ± 0.8*

9.06 ± 1.01 (16)

9.75 ± 0.90 (16)

7.42 ± 1.03 (12)

11.1 ± 0.5 (19)

10.7 ± 0.4 (19)

Proportion of pups born alivea

0.98 ± 0.01

0.99 ± 0.01

0.98 ± 0.02

0.90 ± 0.60*

0.86 ± 0.09 (16)

0.96 ± 0.02 (16)

0.92 ± 0.06 (12)

0.98 ± 0.01 (19)

0.99 ± 0.01 (19)

Live pup weighta

1.65 ± 0.02

1.59 ± 0.03

1.58 ± 0.02

1.52 ± 0.04*

Adjusted live pup weightab

1.65 ± 0.02

1.61 ± 0.02

1.59 ± 0.02*

1.48 ± 0.03*

1.71 ± 0.04 (14)

1.72 ± 0.04 (16)

1.51 ± 0.04 (12)*

1.55 ± 0.02 (19)

1.45 ± 0.02 (19)*

* Significantly different from control group (P < 0.05)

aMean ± SE. Number of observations included in parentheses were appropriate

bMeans adjusted for total number of live and dead pups per litter by analysis of covariance

 

Table 2: Bodyweights, organ weights and sperm parameters of male mice in the continuous breeding study and male mice in the offspring fertility test

Observationa,b

Continuous breeding study

Offspring fertility test

Control

2.5 % EGPE in feed

Control

1.25% EGPE in feed

Number of males

37

20

20

20

Bodyweight (g)

38.81 ± 0.59

36.63 ± 0.58*

34.61 ± 0.73

30.71 ± 0.59*

Organ weights

Liver (g)

2.05 ± 0.03

2.48 ± 0.05*

1.85 ± 0.04

2.05 ± 0.04*

Right epididymis (mg)

64 ± 4

54 ± 6

50 ± 1

43 ± 1

Right testis (mg)

135 ± 3

139 ± 5

124 ± 4

117 ± 4

Seminal vesicles (mg)

470 ± 15

446 ± 21

357 ± 15

306 ± 14*

Prostate (mg)

49 ± 3

45 ± 4

30 ± 4

33 ± 4

Sperm parameters

Sperm concentration (106sperm/g caudal tissue)

633 ± 34

668 ± 32

1054 ± 52

1041 ± 60

Motile sperm (%)

65 ± 4

69 ± 5

55 ± 3

60 ± 1

Abnormal sperm (%)

2.1 ± 0.2

2.6 ± 0.3

4.5 ± 0.4

5.9 ± 1.3

aMean ±SE

bOrgan weights adjusted for bodyweight by analysis of covariance

* Significantly different from control (P < 0.05)

 

Table 3: Bodyweights and liver weights of female mice from the cross mating study and the offspring fertility test.

Observationa,b

Cross mating study

Offspring fertility test

Control

2.5 % EGPE in feed

Control

1.25% EGPE in feed

Number of females

38

17

18

16

Bodyweight (g)

37.1 ± 0.07

36.1 ± 0.9

30.97 ± 0.52

28.91 ± 0.50*

Liver (g)

2.15 ± 0.07

3.44 ± 0.11*

1.81 ± 0.04

2.08 ± 0.05*

aMean ±SE

bOrgan weights adjusted for bodyweight by analysis of covariance

* Significantly different from control (P < 0.05)

 

Table 4: Bodyweights of the offspring from the continuous breeding test (litter 5) at birth, weaning and the beginning of the offspring fertility test

Age (days)

Continuous breeding test

Control

0.25% EGPE in diet

1.25% EGPE in diet

2.5% EGPE in diet

Birth (Day 0)a

Male

1.77 ± 0.03 (34)

1.70 ± 0.04 (15)

1.69 ± 0.07 (18)

1.60 ± 0.04 (12)*

Female

1.68 ± 0.03 (34)

1.66 ± 0.05 (15)

1.66 ± 0.07 (18)

1.52 ± 0.05 (12)*

Weaning (Day 21)b

Male

9.82 ± 0.44 (41)

9.43 ± 0.45 (42)

7.35 ± 0.27 (33)*

5.84 ± 0.22 (32)*

Female

9.80 ± 0.41 (39)

8.34 ± 0.32 (54)

7.56 ± 0.29 (32)*

5.57 ± 0.24 (24)*

Mating trial (Day 74 ± 10)c

Male

35.57 ± 0.87 (20)

34.77 ± 0.70 (20)

30.82 ± 0.62 (20)*

28.76 ± 1.94 (3)*

Female

28.00 ± 0.62 (20)

27.15 ± 0.60 (20)

25.69 ± 0.37 (20)*

24.24 ± 0.82 (3)*

* Significantly different from control group (P < 0.05)

aMean ± SE for male or female pup weight per litter; number of litters evaluated indicated in parentheses

bMean ± SE for individual pup weights; number of pups weighed indicated in parentheses

cMean ± SE for individual mice

 

Conclusions:
Administration of the test substance did not affect the ability to produce 5 consecutive litters in the continuous breeding phase of the study; there was a small decrease in the number of pups/litter and in pup weight in the high dose group, 2.5%. When examined in a crossover study, the affected reproductive component was considered to be from the female, however this was considered inconclusive after assessment, with the only finding being reduced pup weight. Fertility was only minimally compromised, however neonatal toxicity was observed, with increased lethality observed throughout lactation, weaning and puberty. Second generation fertility was found not to be affected, a small decrease in live pup weight was observed. The test substance was found to be toxic to immature mice of both sexes. Under the conditions of the test, the NOAEL for both parental and reproductive toxicity was found to be 400 mg/kg bw/day.
Executive summary:

The reproductive toxicity of the test substance was investigated in Swiss CD-1 mice. Mice were exposed to 2-phenoxyethanol in diet at concentrations of 0, 0.25, 1.25 and 2.5% equivalent to 0, 0.4, 2.0 and 4 g/kg bw/day (a range finding study was conducted with concentrations of 0, 1.25 and 2.5, 5, 7.5 and 10% in feed for 2 weeks). Observations during the study included general toxicity, sperm analysis, reproductive performance, pup abnormalities and assessment of possible sex related effects (crossover mating).

Administration of the test substance did not affect the ability to produce 5 consecutive litters in the continuous breeding phase of the study; there was a small decrease in the number of pups/litter and in pup weight in the high dose group, 2.5%. When examined in a crossover study, the affected reproductive component was considered to be from the female, however this was considered inconclusive after assessment, with the only finding being reduced pup weight. Fertility was only minimally compromised, however neonatal toxicity was observed, with increased lethality observed throughout lactation, weaning and puberty. Second generation fertility was found not to be affected, a small decrease in live pup weight was observed. The test substance was found to be toxic to immature mice of both sexes. Under the conditions of the test, the NOAEL for both parental and reproductive toxicity was found to be 400 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
A two generation reproductive toxicity study with a reliability score of 2 is available and so the quality of the database is sufficient.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Heindel et al (1990) studied the effects of 2-phenoxyethanol in Swiss CD-1 mice in a continuous breeding study. The study was performed in line with GLP according to the continuous breeding methodology which ensured that all stages of the reproductive cycle were encompassed in the test. The study was reported to a high standard and was therefore assigned a reliability score of 2 in accordance with Klimisch (1997). Under the conditions of the test, the test substance was found to have only minimal effects of fertility. A NOAEL of 400 mg/kg bw/day was concluded from the findings of the study.

Nagano et al (1984), investigated the histopathological effects of 2-phenoxyethanol on the testes of male ICR mice. The study was performed in line with good scientific standards, however, the results on 2-phenoxyethanol was reported in an assessment with several other chemicals using three different methods. The information presented on 2-phenoxyethanol was extremely brief, and therefore was assigned a reliability score of 3 in accordance with Klimisch (1997). Under the conditions of the test, the test substance was found not to exhibit any effects on the testes of mice.

Short description of key information:
Key study: Heindel et al (1990), Continuous breeding study with cross-over mating trial and offspring fertility test: Fertility only minimally compromised, NOAEL 400 mg/kg bw/day.

Supporting study: Nagano et al (1984): Testicular toxicity - No effects were observed in male mice dosed with 500, 1000 or 2000 mg/kg bw/day for 5 weeks - 2-phenoxyethanol.

Justification for selection of Effect on fertility via oral route:
Two generation reproductive toxicity study selected on the basis that it is of sufficient reliability to address the data requirement and the supporting study only determined the effect of the test material on mouse testis.

Effects on developmental toxicity

Description of key information
Scortichini et al (1987) -  Equivalent to OECD 414 (Rabbits) - No effects observed at maternally toxic doses of 2-phenoxyethanol. Developmental toxicity/teratogenicity NOAEL 600 mg/kg bw/day, Maternal toxicity NOAEL 300 mg/kg bw/day.
Link to relevant study records
Reference
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.
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
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
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
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.
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.
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.
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:
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.
Abnormalities:
not specified
Developmental effects observed:
not specified

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

 

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
adverse effect observed
600
Quality of whole database:
A pre-natal developmental toxicity study with a reliability score of 2 is available and so the quality of the database is sufficient.
Additional information

Scortichini et al (1987) was performed to a method equivalent to OECD 414. The study selected the rabbit as the test system, as it is known to be the most sensitive to this group of chemicals. The study was performed in line with good scientific principles and reported to a high standard. In accordance with Klimisch (1997), the study was assigned a reliability score of 2, as the study was performed on a read-across substance. Under the conditions of the test, the test material 2-phenoxyethanol was found not to exhibit foetal toxicity up to 600 mg/kg bw/day, at a level which was found to be maternally toxic.


Justification for selection of Effect on developmental toxicity: via dermal route:
Only study available.

Justification for classification or non-classification

According to Regulation EC 1272/2008 and Directive 67/548/EEC, the substance does not meet the criteria for classification as toxic to reproduction in respect to both effects on fertility and also development and teratogenicity.

Additional information