Registration Dossier

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:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The RACB protocol does not possess a defined pre-mating exposure. Especially potential effects on male fertility are less likely to become manifest than in the OECD 416 protocol. This deficiency is in part compensated by the cross-breeding (Task 3) which demonstrated the absence of effects on male fertility after a one-week pre-mating exposure.
Guideline:
other: Reproductive Assessment by Continuous Breeding (RACB); protocol devised by the NTP
Principles of method if other than guideline:
Evaluation of reproductive toxicity of Ethylene Glycol Monophenyl Ether (2-phenoxyethanol) was conducted in Swiss CD-1 mice using the RACB protocol devised by the National Toxicology Program (NTP).
GLP compliance:
yes
Remarks:
Research triangle institute (stated in publication)
Limit test:
no
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding laboratories Inc., NY
- Age at study initiation: 11 weeks of age at the start of the continuous breeding
- Diet (ad libitum)
- Water (ad libitum)
- Housing: group housed in solid bottom polypropylene or polycarbonate cages
- Acclimation period: 5 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 degrees C ±2
- Humidity (%): Not stated in publication.
- Air changes (per hr): Not stated in publication.
- Photoperiod (hrs dark / hrs light): 14/10

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Diet Preperation
EPGE was administered in the feed which was available ad libitum. Each dose was independently blended into a small amount of ground NIH-07 diet (Zeigler Bros, Gardener, PA). The mixture was then added to the pre-weighed portion of the feed and mixed in a patterson kelly 8-quart blender for 15 minutes with the intensifier bar in operation for the first 5 min. Dosed feed was shown to lose about 5% EGPE over 7 days in the cage, therefore dosed feed was prepared fresh weekly. Concentration of EGPE was found to be within 96-105% of the concentration expected within the feed.
Details on mating procedure:
Continuous breeding
- M/F ratio per cage: 1
- Length of cohabitation: 98 days following 7 days of premating dosing with the test article.

The RACB protocol involves a 98-day continuous breeding period, with a 7 day premating dosing period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Aliquots of six representative samples analyzed over the course of the study showed the concentration of EGPE in the preperations to be within 96-105% of the expected values.
Duration of treatment / exposure:
7 day premating and 98 day cohabitation period.
Frequency of treatment:
Administration in oral feed, feed available ad libitum
Details on study schedule:
Task 1: Preliminary dose setting

EGPE was tested at 0.0, 1.0, 2.5, 5.0, 7.5 and 10% in the feed during the 14 day range finding. n = 8/sex/group. Enpoints were clinical signs, body weight and food food consumption.

Task 2: Continous Breeding

The continous breeding phase consisted of a control group (40 breeding pairs), and three dose groups (20 pairs/group). The continous breeding invlolved dosing for 7 days premating and 98 days of cohabitation.

Endpoints observed were: clinical signs, parental body weights, fertility (numbers pairs producing litters/numbers of breeding pairs), litters per pair, lives pups per litter, proportion of pups born alive, sex of live pups, the pup body weights within 18 hours of birth and food and water consumption.

Following the 98 continous breeding period the pairs are seperated and house one animal/cage with continued dosing. Any litter born after the continous breeding are reared by the dam until weaning, after which the chemical is provided at the same concentration as during task 2. The animals are then used for second generation fertility assessment.

Task 3: 1 week mating cross over trial

When a positive effect on fertility is seen during task 2, task 3 is conducted on parental animals after the last litter is weaned to determine the affected sex.

Task 3 consists of three groups of 20 pairs each, control males x control females, control males x high dose females, and control females x high dose males. Pairs are mated for 7 days or until a copulatory plug is detected, whichever is first. Chemical treatment is disconitnued for all animals in this week and then reinstated at the appropriate dose until necropsy.

Endpoints for task 3 are indenitical as task 2. At the end of task 3 F0 males and females are necropsied , the endpoints evaluated are selective organ weight, body weight, epididymal sperm motility, morphology and number, and estrous cyclicity as monitored by vaginal lavage for the preceeding 7 days. Selective organs are examined microscopically after they are fixed in 10% neutral buffered formalin (Bouins fixative for testes), embedded in paraffin and stained with hematoxylin and eosin according to standard procedures.

Task 4: Offspring assessment.

In this phase the last litter from task 2 is nursed, weaned, reared to sexual maturity while housed by sex two or three per cage and exposed to the same concentration of test chemical in feed as their parents. At 74 ± 10 days of age, males and females from different litters within the same treatment group are cohabited for 7 days or until copulatory plug is detected, whichever is less, and then housed singly until delivery. The endpoints for this mating trial are the same as those in task 2. At the end of task 4 the F1 mice are necropsied, the endpoints are the same as the necropsy in F0 mice.

Remarks:
Doses / Concentrations:
0, 0.25, 1.25, 2.5 %
Basis:
nominal in diet
These concentrations produced calculated consumption estimates of approximately 375, 1875 and 3700 mg/kg/day.
No. of animals per sex per dose:
Task 1: 8 animals per sex per group
Task 2: 40 breeding pairs for control, 20 breeding pairs per treatment group
Task 3: 3 groups of 20 pairs
Task 4: 19 pairs/group
Control animals:
yes, concurrent no treatment
Details on study design:
Animals were 6 weeks old upon receipt and were quarantined for 2-5 weeks. During this period, 2 females and 2 males were killed and their sera was analyzed for 11 viruses. All tests were negative. They were randomly assigned to groups by body weight.
Parental animals: Observations and examinations:
Food consumption, parental body weights, mortality, clinical signs of toxicity of parents were evaluated, fertility (number of pairs producing a litter/number of breeding pairs).
Oestrous cyclicity (parental animals):
Yes, examined (estrous cycle length)
Sperm parameters (parental animals):
Testes weight, epididymis weight, enumeration of cauda epididymidis sperm reserve, sperm motility, sperm morphology
Litter observations:
Number and sex of pups, live births, live pup weight (within 18 hr of birth)
Postmortem examinations (parental animals):
Organ weights:
Males: testes, epididymis (total and cauda), prostate, seminal vesicles, kidney w/adrenals, liver.

Females: Liver and kidenys w/adrenals.

Histopathology: No details of histopathology are included within the publication.
Postmortem examinations (offspring):
Organ weights:
Males: testes, epididymis (total and cauda), prostate, seminal vesicles, kidney w/adrenals, liver.

Females: Liver and kidenys w/adrenals.

Histopathology: No details of histopathology are included within the publication.
Statistics:
The Cochran-Armitage test was used to evaluate any dose-related trends in fertility (task 2). A chi square test was used to analyze data from Task 3 to establish overall differences in fertility between groups. Pairwise comparisons between the control and dosed groups were made with the Fisher's exact test. The number of litters and the number of live pups per litter were computed on a fertile pair basis and treatment group means were determined. The proprtion of of live pups was defined as the number of pups born alive , divided by the total number of pups produced by each 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. Dose groups means for these parameters were analyzed using a Kruskal-Wallis test. Ordered differences were tested for by Jonckheere's test. The Wilcoxon-Mann-Whitney U test was used to make intergroup pairwise comparisons.

An analysis of covariance was performed to correct for the potential effect of the number of pups per litter on the average pup weight. The covariate used was average litter size, including live and dead pups. Least squares estimated of dose group means, adjusted for litter size, were computed and tested for overall equality using an F test and pairwise equality was tested using a t test. Average organ weights adjusted for body weight were tested for equality an analysis of covariance. Absolute organ weights were analyzed by the Kruskal-Wallis and Wilcoxon-Mann-Whitney U tests. Dose-related trends were tested for by Jonckheere's test.

Analyses were performed on data for males and females separately and with both sexes combined. The criterion for significance was p<0.05.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not specified
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Task 1

EGPE was tested at 0.0, 1.0, 2.5, 5.0, 7.5 and 10% in the feed for the dose rangefinding. During this 2 week study, animals in the control, 1.0, 2.0 and 5% groups on average gained 16, 15, 12 and 5% respectively in comparison with their initial weight. In the 7.5 and 10% dose groups, both males and females lost 10% of their initial body weight and three of each sex died. Based on these data a dietary level of EGPE selected for task 2 were 0.0, 0.25, 1.25 and 2.5%.

Task 2

EGPE toxicity was seen in F0 males and female. Toxicity was observed in the form of reduction in body weight (males) and increased liver weights (males and females), there was no significant effect on food consumption during the duration of task 2. Average food consumption was approximately 5.6 g/day/mouse, therefore the daily dose of EGPE for males in the 0.25, 1.25 and 2.5% groups was approxiamtely 375, 1875 and 3700 mg/kg respectively, the daily dose in females varied with the stage of gestation due to fluctuations in body weight.

Continuous exposure of CD-1 mice to dietary levels of 0.25, 1.25 and 2.5% did not affect the number of pairs able to produce at least one litter (fertility index). Exposure to 2.5% EGPE tended to reduce the number of litter/pair and significantly reduced litter size and proportion of pups born alive, the high dose group had 19% fewer live pups/litter compared to controls. These affects were not seen in the 0.25 and 1.25% groups. There was also a significant dose related decrease in adjusted live pup weight during the continous exposure of F0 breeding pairs to EGPE with 4 and 10% reduction in the middle and high dose groups repectively. Examination of litter data for the breeding pairs revealed that in the high dose group only 12 out of 26 (60%) pairs had a fifth litter compared to 36 out of 40 (90%) for control pairs. However the number of pups/litter was not different at 9.8 ± 0.6 for the controls versus 8.8 ± 1.1 for the high dose.

Task 3

The proportion of detected matings, pups born alive, or number of pups/litter did not differ significantly across the three combinations of breeding pairs, absolute live pup weight adjust for total pups / litter was significantly decreased for the control males x 2.5 % test substance females versus the control females x control males and 2.5 % test substance males x control females. The results of this crossover study were inconclusive since neither mating nor fertility indices were effected by EGPE pretreatment. Only live pup weight was significantly decreased by 12% in the control x 2.5 % female EGPE mating.

At necropsy 3 weeks after the cross over mating trial there was a signficant decrease in body weight for F0 males (6%) but not for F0 females exposed to 2.5% EGPE in the diet. When adjusted for bodyweight absolute liver weight was significantly elevated in both males and females (14 and 55% respectively). There was no significant differences between control and the 2.5% EGPE males with respect to right testis, prostate and epididymis weights as well as sperm concentration, percentage of motile sperm and percentage of abnormal sperm.

EGPE reduced reproductive performance at doses that increased liver weights in treated F0 mice.

Dose descriptor:
LOAEL
Remarks:
General effects
Effect level:
ca. 3 700 mg/kg bw/day
Sex:
male
Basis for effect level:
other: based on increased liver weight and reduced body weight.
Dose descriptor:
LOAEL
Remarks:
Reproductive effects
Effect level:
> 3 700 mg/kg bw/day
Sex:
male
Basis for effect level:
other: based on lack of effect on male fertility at all dose levels
Dose descriptor:
LOAEL
Remarks:
General effects
Effect level:
ca. 3 700 mg/kg bw/day
Sex:
female
Basis for effect level:
other: based on increased liver weight
Dose descriptor:
LOAEL
Remarks:
Reproductive effects
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
female
Basis for effect level:
other: based on reduced offspring body weight
Dose descriptor:
NOAEL
Remarks:
General effects
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
male
Dose descriptor:
NOAEL
Remarks:
reproductive effects
Effect level:
ca. 3 700 mg/kg bw/day
Sex:
male
Dose descriptor:
NOAEL
Remarks:
general effects
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
female
Dose descriptor:
NOAEL
Remarks:
reproductive effects
Effect level:
ca. 375 mg/kg bw/day
Sex:
female
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
Task 4:

Body weights at birth were reduced in the high dose group (~10% reduction compared to controls). There was reduced body weight gain to weaning: the middle and high dose groups weighed 25 % and 58 % less than controls at weaning on postnatal day 21; on postnatal day 74, the weight differences were 11 % and 17 %, respectively.

Mortality was also increased in the middle and high dose groups from weaning to mating postnatal day 74. Of the 65 pups weaned in the 1.25% group 40 survuved until postnatal day 74. In the high dose group: of the 56 pups weaned only a total of 6 survived to mating at postnatal day 74. This provided too few animals for statistical analysis in the 2.5% group and as such the fertility and reproductive parameters of F1 animals was only assessed in the 1.25% group. At the mating of the second generation there were no statistically significant effects on the proportion of copulatory plug positve mating (mating index), fertile pairs (fertility index), pups born alive or number of pups per litter relative to the F1 controls. As seen with the F0 pairs, live pup weights (F2 pups) were reduced by 7% in the 1.25% group for the F1 pairs. There was also an increase in adjusted liver weight (up 11% in males and 15% in females).

At conclusion of the F1 mating trial the adult mice were necropsied. Body weight was significantly decreased in both males (11%) and females (7%) fed 1.25% groups, while liver weight (adjusted for body weight) was increased compared to the controls. In males at 1.25% absolute testis weight was 16% less and relative seminal vesicle weight was 16% less than controls. There were no effects on right testis, prostate or epdidymal weights or percentage of motile sperm or sperm concentration.

The test substance caused reduced body weight in neonates in Task 2, 3, and 4, and reduced post-natal survival in F1 animals that were raised to sexual maturity.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
male
Basis for effect level:
other: based on reduced body weight, increased liver weight, decreased testes weight and increased lethality in pups during lactation.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
female
Basis for effect level:
other: based on reduced pup weight and increased lethality in pups during lactation.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 375 mg/kg bw/day
Sex:
male/female
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
ca. 375 mg/kg bw/day
Sex:
male/female
Reproductive effects observed:
not specified
Conclusions:
2-phenoxyethanol produced reproductive and developmental toxicity at doses that increased liver weight in F0 and F1 mice. 2-phenoxyethanol caused toxicity in growing animals as evidenced by the reduced body weight in neonates in tasks 2, 3 and 4, and the large increase in postnatal lethality as the F1 animals grew to the age of mating.
Executive summary:

In summary 2-phenoxyethanol produced reproductive and developmental toxicity at doses that increased liver weight in F0 and F1 mice. 2-phenoxyethanol caused toxicity in growing animals as evidenced by the reduced body weight in neonates in tasks 2, 3 and 4, and the large increase in postnatal lethality as the F1 animals grew to the age of mating. From this study it was not possible to determine if the increased lethality seen in offspring was caused by quality of milk, lactation problems or EGPE transferred in the milk, however because F1 animals continued to die after weaning it suggests that immature mice are more sensitive to EGPE than the adult.

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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
375 mg/kg bw/day
Study duration:
subchronic
Species:
mouse
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The registration substance is a monomer in an imported polymer and the lifecycle of this monomer ends outside of the EU. The concentration of unbound monomer in the imported polymer is 0.05% (w/w). This polymer is formulated (diluted) further into finished lubricant oils, in which the levels of unbound monomer will be considerably lower than 0.05% (typically 0.0005% in a finished lubricant oil). Data has been generated up to the sub-acute level for the registration substance and has shown no significant toxicological hazards except eye irritancy (H319: Causes serious eye irritation). It is considered that with such low concentrations of unbound monomer in the polymer/finished oil, the level of human exposure will be very low and as such the hazard posed by the unbound monomer is negligible. Importantly, it has been established through conduct of an in vivo micronucleus study in rat that the registration substance does not have mutagenic potential, ruling any potential non-threshold hazard. Therefore, it can be concluded with confidence that the low levels of unbound registration substance in the imported polymer pose a negligible hazard to human health.

Based on the low potential for human exposure and therefore low risk to human health, no further in vivo studies are proposed for the registration substance. Instead read-across will be used to an analogous substance (2 -phenoxyethanol) in order to fill the remaining Annex VIII and IX data gaps. The target substance 2-(2-naphthyloxy)ethanol and the source substance 2-phenoxyethanol are structurally similar substances that possess similar physical-chemical characteristics and as such are expected to possess similar toxicokinetic profiles. The substances have almost identical toxicological profiles up to the sub-acute level. Given the information available it is evident that data generated for 2-phenoxyethanol reflects the toxicological profile of 2-(2-naphthyloxy)ethanol, as such read-across between the two substances is acceptable. It is considered that reproductive and developmental toxicity data generated for 2-phenoxyethanol can be used as read-across to 2-(2-naphthyloxy)ethanol as similar findings would be expected. Full justification for use of read-across between 2-phenoxyethanol and 2-(2-naphthyloxy)ethanol is attached to section 13 of this dataset.

It is considered that 2-Phenoxyethanol does not show evidence of having intrinsic specific properties to produce an adverse effect on reproduction and development. In the continuous breeding study, effects on reproduction and development were observed, but only at doses that were maternally toxic. It is believed that the effects on reproduction and development are non-specific and of a secondary consequence of other toxic effects. At doses of 375 mg/kg bw/day no maternal toxicity was observed nor were any reproductive or developmental effects.

Fertility was only minimally compromised and again this only occurred at high doses that were toxic to the F0 animals. It was not possible from the study to determine the cause of the reduced body weight and increased lethality in pups during lactation, weaning and puberty, however as mice continued to die after weaning it suggests that the immature mouse is more sensitive to 2-Phenoxyethanol. It is important to note that the high doses used within the continuous breeding study were greater than the OECD-recommended limit dose of 1000 mg/kg bw/day for repeated dose studies. In summary the reproductive toxicity of 2 -phenoxyethanol was only evident in females and occurred at doses that were maternally toxic. 2 -phenoxyehtanol was also notably toxic to immature mice of both sexes at 1875 and 3700 mg/kg bw/day.

The data presented for material 2 -phenoxyethanol (EC 204-589-7) is considered appropriate for read-across to the registration substance (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.


Short description of key information:
NOAEL for reproductive effects = ca.375 mg/kg bw/day).

Justification for selection of Effect on fertility via oral route:
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.

Effects on developmental toxicity

Description of key information
The NOAEL for developmental toxicity was 600 mg/kg bw/day when 2-phenoxyethanol was applied dermally to New Zealand white rabbits. Maternal toxicity was seen at 1000 mg/kg bw/day and 600 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:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was comparable to a guideline study with acceptable restrictions.
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
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.
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
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.

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.
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.
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:
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.
Abnormalities:
not specified
Developmental effects observed:
not specified
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.

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
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Additional information

The registration substance is a monomer in an imported polymer and the lifecycle of this monomer ends outside of the EU. The concentration of unbound monomer in the imported polymer is 0.05% (w/w). This polymer is formulated (diluted) further into finished lubricant oils, in which the levels of unbound monomer will be considerably lower than 0.05% (typically 0.0005% in a finished lubricant oil). Data has been generated up to the sub-acute level for the registration substance and has shown no significant toxicological hazards except eye irritancy (H319: Causes serious eye irritation). It is considered that with such low concentrations of unbound monomer in the polymer/finished oil, the level of human exposure will be very low and as such the hazard posed by the unbound monomer is negligible. Importantly, it has been established through conduct of an in vivo micronucleus study in rat that the registration substance does not have mutagenic potential, ruling any potential non-threshold hazard. Therefore, it can be concluded with confidence that the low levels of unbound registration substance in the imported polymer pose a negligible hazard to human health.

Based on the low potential for human exposure and therefore low risk to human health, no further in vivo studies are proposed for the registration substance. Instead read-across will be used to an analogous substance (2 -phenoxyethanol) in order to fill the remaining Annex VIII and IX data gaps. The target substance 2-(2-naphthyloxy)ethanol and the source substance2-phenoxyethanol are structurally similar substances that possess similar physical-chemical characteristics and as such are expected to possess similar toxicokinetic profiles. The substances have almost identical toxicological profiles up to the sub-acute level.Given the information available it is evident that data generated for 2-phenoxyethanolreflects the toxicological profile of 2-(2-naphthyloxy)ethanol, as such read-across between the two substances is acceptable. It is considered that reproductive and developmental toxicity data generated for2-phenoxyethanolcan be used as read-across to 2-(2-naphthyloxy)ethanol as similar findings would be expected. Full justification for use of read-across between 2-phenoxyethanol and 2-(2-naphthyloxyethanol) is attached to section 13 of this dataset.

Results of a developmental toxicity study in New Zealand white rabbits indicate 2-Phenoxyethanol is not a teratogen or developmental toxicant when administered dermally from gestation days 6 to 18 at doses of 300, 600 and 1000 mg/kg bw/day. Both 1000 and 600 mg/kg bw/day showed maternal toxicity. A NOAEL for developmental toxicity of 600 mg/kg bw/day was derived as no effects on the fetuses were seen despite the toxicity in the mother being apparent at this dose. No effects were seen in the fetuses of the 5 surviving animals at 1000 mg/kg bw/day animals, however this dose could not be used to determine the NOAEL as insufficient parent animals survived to render the results statistically significant.

It is concluded that a dermal study was appropriate given that dermal exposure is a likely route of exposure. Furthermore clear maternal toxicity was seen in the rabbits at both medium and high dose levels indicating that the material is getting absorbed through the skin and entering systemic circulation.

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.


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

Justification for classification or non-classification

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

It is considered that 2-Phenoxyethanol does not show evidence of having intrinsic specific properties to produce an adverse effect on reproduction and development. In the continuous breeding study, effects on reproduction and development were observed, but only at doses that were maternally toxic. It is believed that the effects on reproduction and development are non-specific and of a secondary consequence of other toxic effects. At doses of 375 mg/kg bw/day no maternal toxicity was observed nor were any reproductive or developmental.

Fertility was only minimally compromised and again this only occurred at high doses that were toxic to the F0 animals. It was not possible from the study to determine the cause of the reduced body weight and increased lethality in pups during lactation, weaning and puberty, however as mice continued to die after weaning it suggests that the immature mouse is more sensitive to 2-Phenoxyethanol. It is important to note that the high doses used within the continuous breeding study were greater than the OECD-recommended limit dose of 1000 mg/kg bw/day for repeated dose studies. In summary the reproductive toxicity of 2 -phenoxyethanol was only evident in females and occurred at doses that were maternally toxic. 2 -phenoxyehtanol was also notably toxic to immature mice of both sexes at 1875 and 3700 mg/kg bw/day.

Fetotoxicity and teratogenic potential of 2-Phenoxyethanol was assessed by dermally exposing groups of 25 pregnant rabbits on gestation days 6-18 at 0, 300, 600 and 1000 mg/kg bw/day. Maternal toxicity was observed at 600 and 1000 mg/kg/day in the form of intravascular haemolysis of red blood cells and death in some animals. Nine rabbits in the 1000 mg/kg/day dose and 5 rabbits in the 600 mg/kg/day dose groups died or were sacrificedin extremis.Insufficient parent rabbits survived until gestation day 28 in the 1000 mg/kg/day dose group, for this dose level to be used to generate an NOAEL despite the absence of any affects seen in the foetuses of the surviving animals. Examination of foetuses indicated 2-Phenoxyethanol was not embryotoxic, fetotoxic or teratogenic at any doses used, even those that caused maternal toxicity. A NOAEL for developmental toxicity of 600 mg/kg/day was derived for this study.

Taking both of these key studies into account it can be concluded that 2-Phenoxyethanol does not  show evidence of having intrinsic specific properties to produce an adverse effect on reproduction and development. In the continuous breeding study effects on reproduction were only seen at doses that were maternally toxic and were considered non-specific and of a secondary consequence of other toxic effects. And in the teratogenicity/fetotoxicity study no effects on development are seen even at doses that are maternally toxic.

Based on these data, classification of 2-phenoxyethanol is not required for reproductive and developmental toxicity. This is in line with the conclusions on classification as presented in the REACH registration dossier for 2-phenoxyethanol available on the ECHA dissemination website. 2-phenoxyethanol and 2-(2-naphthyloxyethanol) are expected to have similar toxicological profiles due to their structural similarities, therefore the reproductive and developmental data generated for 2-phenoxyethanol will be read-across to 2-(2 -naphthyloxyethanol) for the purposes of hazard classification. Based on this read-across, it is concluded that the registration substance does not require classification for reproductive or developmental toxicity.