Registration Dossier

Administrative data

Description of key information

An OECD 422 repeated dose/reproductive screening study using Di-EPh; Several repeated dose oral, inhalation and dermal toxicity studies have been performed using phenoxyethanol (PE). The benchmark dose method was used to derive a BMDL10. The most critical effect was determined to be the renal hyperplasia in male rats. Combining the subchronic and chronic studies in rats a BMDL10 of 369 mg/kg bw/day has been derived.

In a 90 -day repeated-dose dermal toxicity study in white rabbits toxicologically non relevant effects were observed. Therefore the highest dose tested (500mg/kg bw/day) was designated as the NOAEL for systemic toxicity.

In a 14 -day inhalation study with rats pathological examinations revealed no treatment-related changes in either males or females. Morphological changes indicating irritation were found in nasal cavity, larynx, and lung of male and female mid- and high-concentration animals. A NOAEC of 48.2 mg/m3 was determined.

The values being used for CSA have been corrected for the molecular weight difference between the target (Di-EPh) and source (EPh) chemicals (182.2/138.2) = 1.32 molecular weight correction factor.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed in accordance with OECD guidelines and GLP. Data from reliable source (Japanese Ministry of Health, Labour and Welfare). No English test report is available (study in Japanese), but an English summary and all tables and figures in English are available.
Justification for type of information:
Read Across to an analogue based on structural similarity. An analogue justification is attached to section 13 of the dataset.
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: the average weight of males was 126 g and 99 g of the females
Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability was confirmed using quantitative HPLC analysis
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily, the test substance was administered to the drinking water.
Remarks:
Doses / Concentrations:
0, 96, 185, 369, 687, and 1514 mg/kg/day in males and 163, 313, 652, 1000, and 1702 mg/kg/day in females
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 1250, 2500, 5000, 10000, and 20000 mg/L
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION, WATER CONSUMPTION AND BODY WEIGHT:
- Time schedule: weekly

HAEMATOLOGY:
- Time schedule for collection of blood: The surviving animals were bled at the end of the study.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: Haematology was examined in every male rat from all dose groups. Haematology in female rats was examined in all rats from all dose groups with the exception from 1 rat in the control group.
- Parameters: Red blood cell count, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration, platelet count, reticulocytes, prothrombin time, activated partial thromboplastin, white blood cell count, and differential white blood cell count.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: The surviving animals were bled at the end of the study.
- Animals fasted: No data
- How many animals: All animals
- Parameters: Plasma chemistry analyses included total protein, albumin, A/G ratio, total bilirubin, glucose, total cholesterol, triglyceride, phospholipid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyl transpeptidase, creatine phosphokinase, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, and inorganic phosphorus.

URINALYSIS:
- Time schedule for collection of urine: Urine samples were collected for urinalyses at the end of treatment.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters: pH, protein, glucose, ketone body, bilirubin, occult blood, and urobilinogen
Sacrifice and pathology:
GROSS PATHOLOGY AND HISTOPATHOLOGY: Yes, gross pathology at necropsy, organ weights (thymus, adrenal, testes, ovary, heart, lung, kidney, spleen, liver, brain, and thyroid) and histopathology were conducted.
Statistics:
Incidences of non-neoplastic lesions and urinalysis were analyzed by Chi-square test. Changes in body weight, water consumption, food consumption, hematological and blood biochemical parameters, and organ weights were analyzed by Dunnett’s test.
Details on results:
CLINICAL SIGNS AND MORTALITY
Soiled fur around genitalia was observed in all animals administered with 20,000 mg/L test substance and in a few animals exposed to 5000 and 10,000 mg/L. One male rat in the 20000 mg/L dose group did not survive the 13th study week, all other animals survived until necropsy.

BODY WEIGHT AND WEIGHT GAIN
After 13 weeks a significant decrease in body weight was observed in both sexes of the 20,000 mg/L dose group (±19% lower compared to the control group). Decrease in body weight was also observed in females administered with 10,000 mg/L test substance (±8% lower compared to the control group)

FOOD CONSUMPTION AND WATER CONSUMPTION
During the entire study a decrease in food consumption was observed in males and females in the 20,000 mg/L dose groups (±20% relative to controls) and in females in the 10,000 mg/L dose group (±10% relative to controls). During the entire study water consumption was decreased in females administered with 20,000 mg/L. The decrease in females was approximately 30-40%. In males dosed with 5000, 10000, and 20000 mg/L decreased water consumption was observed untill the 9th week of the study.

HAEMATOLOGY
Red blood cell count and platelet count were reduced in the 10000 and 20000 mg/L dose groups of both sexes. In males platelet count was also reduced in animals exposed to 5000 mg/L. The reduction of platelets in the 5000 mg/L dose group was less then 10% compared to controls and no changes were observed in coagulation parameters. Therefore no evidence of a functional effect was present. In the 10000 and 20000 mg/L dose groups in females and in the 20000 mg/L dose group in males haemoglobin was reduced. MCV and MCH were increased in males (10000 and 20000 mg/L) and females (20000 mg/L). In females administered with 20000 mg/L an increased reticulocyte count was measured.

CLINICAL CHEMISTRY
In males exposed to 20000 mg/L test substance a reduction in total protein, glucose, sodium, calcium and an increase in A/G ratio, total cholesterol, phospholipids, urea nitrogen, potassium was observed. In the 10000 mg/L dose group only the effects on total protein, cholesterol, phospholipids, sodium and potassium were present. No effects were observed at lower dose levels. In female exposed to 20000 mg/L increases in A/G ratio, ALP, and urea nitrogen was observed. In the 10000 mg/L dose group total protein was decreased and urea nitrogen was increased.

URINALYSIS
Statisticaly significant decrease in pH was observed after administration of 20000 mg/L in both sexes. This could be the result of excretion of 2-phenoxyacetic acid, the major metabolite of the test substance.

ORGAN WEIGHTS
In the 10000 mg/L dose groups, the relative liver weight was increased in males and females. In addition in females absolute adrenal weight was decreased and relative kidney and brain weights were increased. In males of the 20000 mg/L dose group a decrease in absolute thymus, testes, heart, lung, and spleen was observed. In addition an increase in relative testes, lung, kidney, liver, brain, and thyroid was determined. In females of the 20000 mg/L dose group decreases in absolute thymus, adrenals, ovaries, heart, lungs, and spleen weights were determined. Relative kidney, liver, and brain weights were increased.

HISTOPATHOLOGY
In 2 males and 1 female dosed with 10000 mg/L, slight urothelial hyperplasia of the renal pelvis was observed. Slight to moderate urothelial hyperplasia was observed in 6 males of the highest dose group. In 2 and 7 females dosed with 10000 and 20000 mg/L test substance, respectively slight to moderate urinary bladder transitional epithelial hyperplasia was observed. Slight urinary bladder transitional epithelial hyperplasia was also observed in one male of the high dose group.

ANALYTICAL DATA DRINKING WATER:
Analytical data verified that the test material content in drinking water was within acceptable limits of the target concentrations (101-102%).
Dose descriptor:
NOAEL
Effect level:
369 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Decreased red blood cells and haemogobin; increased MCV and MCH; increased incidence of slight to moderate hyperplasia in the kidney and urinary bladder.
Critical effects observed:
not specified
Endpoint conclusion
Dose descriptor:
BMDL10
486.5 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
good
System:
other: urogenital
Organ:
kidney

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
06 Sep 2005 - 17 Feb 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 412
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Age at study initiation: about 7 weeks
- Weight at study initiation: males: ca. 227 g, females: ca. 163 g
- Housing: animals were housed singly in makrolon-wire cages (type MD III, Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm²)
- Diet (e.g. ad libitum): milled mouse/rat laboratory diet “GLP”, (Provimi Kliba SA, Kaiseraugst, Basel Switzerland)
- Water (e.g. ad libitum): tap water
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 06 Sep 2005 To: 28 Sep 2005
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
other: air
Remarks on MMAD:
MMAD / GSD: see "Details on inhalation exposure"
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Generator systems:
- Continuous infusion pumps PERFUSOR (B. Braun) for test group 1
- Piston metering pumps (Sarstedt DESAGA) for test group 2 and 3
- Two-component atomizers (stainless steel, Schlick mod. 970)
- Glass mixing stages (BASF)
- Glass cyclonic separators (BASF)


Generation procedure:
For each concentration the test substance was supplied to a two-component atomizer at a constant rate by means of a metering pump. The aerosol was generated with compressed air in a mixing stage with conditioned dilution air and passed via the cyclonic separator into the inhalation system. Due to the vapor pressure of the test substance, in all test groups mixtures of vapor and liquid aerosol are tested. The theoretical vapor concentration is up to 40 mg/m3.


TEST ATMOSPHERE (if not tabulated)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.)
The target concentration of 40 mg/m3 in test group 1 was the saturation vapor concentration. At this concentration, part of the test substance might condensate in the atmosphere, thus liquid aerosols might be formed. To demonstrate the aerosol formation, two cascade impactor measurements were carried out. These resulted in MMADs between 2.9 and 3.7 μm with GSDs of 4.3 and 4.5. The amount of test substance that was trapped by the cascade impactor was approximately 20 % of that measured in the atmosphere, which was in the expected range for condensation effect. Due to the long sampling time (150 min), the condensation on the cascade stages during the sampling period might be much more pronounced in this group than in the other groups. Therefore, the particle size measurements in test group 1 were considered not to reflect the real particle size distribution, but to describe the liquid aerosol formation in the test atmosphere.
Cascade impactor measurements in the test groups 2 and 3 resulted in MMADs between 0.5 and 1.3 μm that were well within the respirable range. The calculated mass fractions of particles below 3 μm aerodynamic size ranged between 72.0 and 85.2%. The EACD (effective aerodynamic cut-off diameter 50%) of the last impactor stage is 1.2 μm. MMAD values below 1.2 μm are gained by extrapolation outside the effective measuring range of the impactor. Therefore the real value may lie between the calculated one and 1.2 μm.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days per week for 14 days (10 exposures)
Remarks:
Doses / Concentrations:
0, 40, 200, 1000 mg/m³
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice each working day and once on weekends and holidays
- Cage side observations checked: evident signs of toxicity or mortality


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On exposure days clinical examination was performed before, during and after exposure.


BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the animals was determined at the start of the preflow (day-2), at the start of the exposure period (day 0) and then on day 7 as well as one day prior to gross necropsy (day 13).


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of the study (with EDTA-K3 as anticoagulant)
- Anaesthetic used for blood collection: Yes (Isoflurane (Essex GmbH Munich, Germany))
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, reticulocytes, prothrombin time (Hepato Quick's test)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the study
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-γ-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, at termination of the study.
Organ weights: liver, kidneys, adrenals, testes, thymus, spleen and lung
HISTOPATHOLOGY: Yes, at termination of the study.

Gross pathological examination was accompanied by the histopathological examination of following selected organs: nasal cavities, larynx, trachea, lungs, mediastinal lymph nodes, thymus, liver, kidneys, spleen, adrenal glands, heart, stomach (fore- and glandular stomach), and esophagus.
Details on results:
In none of the test groups clinical signs of toxicity were observed. Treatment related influence on body weight development or food consumption was found in the high concentration group on study day 7. Clinical pathology examinations revealed no treatment-related changes in either males or females. 14 days of aerosol inhalation of the test substance led to histopathologic findings in the respiratory tract. The respiratory epithelium in the nasal cavity was the target tissue in high and mid concentration males and females. In the very anterior part signs of degeneration and metaplasia were noted, whereas in the more posterior parts hyperplasia was observed. Inflammatory cell infiltrates were seen in the mid and top concentration groups as a reaction upon treatment. These findings are thought to be substance-related and the top concentration groups were slightly more affected. In the larynx (level I) in the area of the very sensitive epithelium at the base of the epiglottis, three male animals of the high concentration group showed a minimal to slight hyperplasia of the respiratory epithelium. This finding is also considered to be substance-related. According o the 1st International ESTP expert workshop --“Larynx squamous metaplasia”. A re-consideration of morphology and meaning in rodent studies and its relevance for humans - held on November 13-14, 2006, at Fraunhofer Institute ITEM Hannover, Germany, minimal, focal epithelial changes of the larynx epithelium predominantly occurring at the base of the epiglottis should be given the descriptive term of an “epithelial alteration” as the morphological criteria of a “laryngeal squamous metaplasia” are not completely met. Those lesions are regarded as “adaptive” and non-adverse in character. Further, inhalative exposure to compounds that do not exert systemic or local genotoxic effects may cause “laryngeal squamous metaplasia”, but no tumor formation is reported in long-term studies known to the workshop experts. For non-genotoxic-compounds, the workshop experts do not regard “laryngeal squamous metaplasia” as a precancerous lesion. The workshop outcome from the ESTP meeting has meanwhile be published in a peer-reviewed journal (Kaufmann W. et al. (2009). Experimental and Toxicological Pathology 61, 591-603).
In males of the mid and top concentration the absolute lung weight (up to 20.4%) and in males of the top concentration the relative lung weight (up to 19.3%) were statistically significantly increased. This is regarded as a substance-related finding although there was no histopathologic correlate that could explain this increased weight. In the lungs there was an increase in thickness of small and terminal bronchi and increased numbers of mucous cells in larger bronchi of mid and top concentration males and females. This was also regarded to be a substance-related finding and is judged to be most likely adaptive. In conclusion, the upper respiratory tract showed mild signs of reaction (degeneration, metaplasia, inflammatory cell infiltration) to the test substance; most sensitive seemed to be the respiratory epithelium of the nose, especially in the anterior septum area. The epithelial lining other sites, e.g. respiratory epithelium of lungs, transitional epithelium larynx was also affected but appeared to be less sensitive.
Clinical pathological examinations revealed no treatment-related changes in either males or females. Morphological changes indicating irritation potential of the test compound were found in nasal cavity, larynx and lung of male and female mid- and high concentration animals. In forestomach no morphological changes were noted.
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
48.2 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: histopathologic findings in the upper respiratory tract (target organ), increased lung weights in males only
Dose descriptor:
LOAEC
Effect level:
246 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: body weight gain and food consumption
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
63.6 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
ok

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
23 Oct 1985 - 08 Oct 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study.
Justification for type of information:
Read Across to an analogue based on structural similarity. An analogue justification is attached to section 13 of the dataset.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Principles of method if other than guideline:
No guideline mentioned in the report.
However, study is equivalent or similar to OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study) with minor deviations regarding weight at study initiation:
Males: about 3250 g; Females: about 3380 g
GLP compliance:
yes
Remarks:
according to FDA and EPA GLP Practice regulation
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazleton-Dutchland, Inc. Denver, Pennsylvania
- Age at study initiation: approx. 5-6 months
- Weight at study initiation: Males: about 3250 g; Females: about 3380 g
- Fasting period before study: none
- Housing: single
- Diet (e.g. ad libitum): 4 oz./day/animal (Certified Laboratory Rabbit Chow #5322, Ralston Purina Co., St. Louis, Missouri)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 21 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 20
- Humidity (%): approx. 50
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: 10 x 15 cm on the back of each test animal.
- Type of wrap if used: Occlusive bandage of absorbent gauze and nonabsorbent cotton held in place using a Lycra/spandex jacket.
- Time intervals for shavings or clipplings: clipping periodically as required during the course of the study.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Dosing volume was approximately 0.05 to 0.50 ml/kg bw/day and was adjusted weekly based on body weight. Control rabbits received 0.5 ml/kg bw/day distilled water
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours per day, 5 days/week for 13 consecutive weeks
Remarks:
Doses / Concentrations:
0, 50, 150, 500 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Results from previous studies
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined daily for general state of health and clinical symptoms of toxicity.


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: The condition of the skin was assessed and recorded prior to each daily dose for the first two weeks and approx. weekly thereafter using a modified Draize scoring system (see attached background material).


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION: Yes, periodically (data were not presented in the report)


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At end of study. In-situ examination of the eyes by a glass slide technique with fluorescent illumination
- Dose groups that were examined: All animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1 week prior to exposure (pre-exposure), 4 week interim, and at test termination.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All animals
- Parameters checked in table were examined.


CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood: 1 week prior to exposure (pre-exposure), 4 week interim, and at test termination
- Animals fasted: No
- How many animals: All animals
- Parameters checked in table were examined.


URINALYSIS: No
Sacrifice and pathology:
GROSS PATHOLOGY: A complete set of organs was collected from each animal and was preserved in neutral phosphate-buffered 10% formalin. The lungs were infused with formalin to their approximate normal inspiratory volume whereas the nasal cavity was flushed with formalin via the pharyngeal duct to ensure rapid fixation of the tissues.

HISTOPATHOLOGY: A complete histopathological examination of tissues was conducted for the rabbits of the control and the high dose groups. For this purpose, tissues were prepared by conventional techniques, stained with Hematoxylin/Eosin and were evaluated by means of light microscopy.
Statistics:
Body and organ weights, as well as hematological and clinical-chemical data were analyzed using Bartlett's test for equality of variances. Based on the outcome, a parametric or nonparametric analysis of variance was conducted, followed by Dunnett's test or the Wilcoxon rank-sum test with Bonferroni's correction. The critical level of significance was p < 0.05. Statistical outliers were identified by a sequential outlier test but were not excluded from analysis except for body weight.
Level of significance: see attached background material
Details on results:
CLINICAL SIGNS AND MORTALITY
No overt signs of systemic toxicity
no mortalities at any dose


BODY WEIGHT AND WEIGHT GAIN
The mean body weights of male and female rabbits were not affected by treatment with 2-phenoxyethanol during the course of the study.


FOOD CONSUMPTION
Results on food consumption were not reported.


OPHTHALMOSCOPIC EXAMINATION
No findings were reported.


HAEMATOLOGY
No treatment-related effects could be evidenced.


CLINICAL CHEMISTRY
No treatment-related effects could be evidenced.
In fact, a statistically significant decrease in AST activity was reported for the females of the 150 mg/kg/d dose group after 4 weeks of treatment, whereas the males of the 50 mg/kg bw /d showed an increased ALT activity at study termination. However, these effects neither showed a dose-response relationship nor were they seen at the other sampling time points. Therefore, these effects were not considered to be treatment-related.


URINALYSIS
Not examined


ORGAN WEIGHTS
No toxicologically significant changes were observed in terminal body weights or absolute and relative organ weights. A statistical decrease in absolute liver weight was identified in female rabbits administered 50 mg/kg bw/day of test substance, but there was no dose-response relationship. Therefore, the statistically identified difference in liver weight was not considered to be treatment-related.


GROSS PATHOLOGY
There were no treatment-related gross pathological changes observed in either male or female rabbits. The few observations in these animals at necropsy were considered spontaneous changes typical of those expected in New Zealand White rabbits of this age. Likewise, there were no treatment-related lesions observed.


HISTOPATHOLOGY: NON-NEOPLASTIC
See attached background material


OTHER FINDINGS
The only potentially treatment-related effects were the sporadic occurrence of erythema (4 males) and the very slight to slight scaling of the skin at the application site (8 males and 9 females at week 7) in animals exposed to 500 mg/kg bw/day. Exfoliation was resolved in all but 2 females during weeks 9-13. As these observations were not associated with any gross or histological changes in the skin, they were not considered to be of toxicological significance.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
LOAEL
Effect level:
> 500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified

No overt signs of toxicity were noted in the animals. There were no deaths. There was no significant effect of treatment on body or organ weights, clinical chemistries, hematologic variables, or pathology. The only potentially treatment-related effects was the sporadic observation of erythema (4 males) and very slight to slight scaling of the skin at the site of test material application (8 males and 9 females at week 7) in animals exposed to 500 mg/kg bw/day.
Exfoliation was resolved in all but 2 females during weeks 9-13. As these observations were not associated with any gross or histological changes in the skin, they were not considered to be of toxicologic significance.

For tables of results refer to attached background material

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
659.2 mg/kg bw/day
Study duration:
subchronic
Species:
rabbit
Quality of whole database:
good

Additional information

Data on Di-EPh

An oral (dietary), OECD 422 study in rats is available for Di-EPh.

There were no effects on body weight, body weight gain, or feed consumption in males or females in the 100 or 300 mg/kg bw/day dose groups and in the males of the 1000 mg/kg bw/day group. During gestation, the high dose females had decreased bodyweight gain and food consumption. Although not statistically significant this effect was considered to be an adverse, treatment related effect.

There were no adverse treatment effects on in life parameters (sensory, motor activity, rectal temperature, grip performance) in any group. Hematological parameters were unaffected by treatments and the only treatment-related effect on clinical pathology parameters was a slightly higher serum urea nitrogen concentration in males and females given 1000 mg/kg/day. This effect was considered to be non-adverse because there were no corresponding histopathologic alterations in the urinary tract, and no evidence of other predisposing conditions such as dehydration or increased protein catabolism.

Very slight hypertrophy of centrilobular and midzonal hepatocytes occurred in males and females given 1000 mg/kg/day. The hypertrophy corresponded with higher absolute and relative liver weights at this dose level in both sexes. These changes were considered to be an adaptive response associated with increased hepatic metabolism of diethylene glycol mono phenyl ether (refer to toxicokinetics section for metabolism information). Males and females given 1000 mg/kg/day had lower incidences of hepatocellular vacuolization (consistent with fatty change), which was interpreted to be a non-adverse effect of treatment. Females given 1000 mg/kg/day had lower absolute and relative thymus weights, which were interpreted to be non-adverse because there were no corresponding histopathologic observations.

There were no treatment-related effects of Di-EPh on neurological or reproductive function, or prenatal neonatal growth and survival of the offspring. Based on these results, the no-observed-effect level (NOEL) for general toxicity was 300 mg/kg/day in both sexes. The n no observed adverse effect level (NOAEL) for males was 1000 mg/kg bw, the highest dose level tested. The critical effect driving the NOEL in the females is a decrease in bodyweight gain and food consumption during gestation. This effect only appeared during gestation and is consistent with the observations in the oral, rat developmental toxicity study on EPh (see section 7.8.2). When considering the 'general toxicity' in the females prior to pregnancy, the NOEL for bodyweight gain and feed consumption is the highest dose of 1000 mg/kg bw/day.

The data from this study indicate Di-EPh has a low order of toxicity. In order to address the REACH requirements for a 90-day repeated dose study, data on the analogue EPh have been utilised. The justification for the use of read across is contained in the document attached at section 13.

Data on EPh

Oral:

Sub-Chronic repeated dose studies:

Two repeated dose drinking water studies (13 weeks) were performed according to OECD 408 and in compliance with GLP. The first drinking water study was conducted with F344/DuCrj rats (JBRC.0459, 2003). 10 rats per sex were exposed to 0, 1250, 2500, 5000, 10000 and 20000 mg/L. Analytical concentrations in drinking water were determined with HPLC. Based on chemical intake data the mean intake of test substance across the duration of the study was estimated to be 96, 185, 369, 687, and 1514 mg/kg/day in males and 163, 313, 652, 1000, and 1702 mg/kg/day in females. Food intake, water intake and body weight were determined weekly. After 13 weeks urinalysis, haematology, blood chemistry, gross pathology, organ weights and histopathology were examined. Effects on the red blood cell parameters and changes in histopathology of the kidney and bladder were observed in rats dosed with 10000 mg/L test substance and above. Based on these results the NOAEL was determined to be 5000 mg/L. This corresponds with an intake of 369 mg/kg/day in males and 652 mg/kg/day in females.

 A similar study was conducted in Cru: BDF1 mice (OECD 408) (JBRC 0460, 2003). The study design and examination/observations were similar to the study in rats. However the dose levels differed and were 0, 1250, 2500, 5000, 10000 and 20000 mg/L in drinking water. The mean intake of test substance across the duration of the study was estimated to be 182, 390, 765, 1178, and 2135 mg/kg/day in males and 236, 478, 948, 1514, and 2483 mg/kg/day in females. Effects on red blood cell parameters (Females: hemoglobin, MCHC, MCV and males: reticulocytes) were found in mice dosed with 20000 mg/L test substance. In males dosed with ≥5000 mg/L changes in blood chemistry parameters (decreases in cholesterol and phospholipids) were measured and suggest an effect on the liver however no histopathological changes were observed in the liver. Based on these effects the NOAEL was determined to be 2500 mg/L and this corresponds to an intake of 390 mg/kg/day.

2-Phenoxyethanol was administered via the diet to Wistar rats in doses of 0, 500, 2500 and 10000 ppm for a period of 13 weeks (Bayer AG, 2002). Additional groups were treated with 0 and 10000 ppm in the same way and observed for a subsequent treatment-free period of 4 weeks for recovery. Satellite groups comprising also recovery groups were treated with doses of 0, 500, 2500 and 10000 ppm for 13-14 weeks or 0 and 10000 ppm, respectively. Animals of all satellite groups were subjected to whole-body-perfusion during necropsy for possible neuropathological investigations. However, since the functional observation battery, motor activity determination and reflex testing did not indicate any treatment related effects, no further neuropathological examinations were performed. Survival was unaffected by 2-phenoxyethanol treatment. Furthermore, there were no treatment-related effects on behaviour, body weight/body weight gains, feed/water consumption, haematological or clinical chemistry parameters, organ weights, or gross or histopathological findings. Some organ weight changes were seen in the treated and/or recovery groups, but there was no consistent pattern of effects and no corresponding histopathology changes; therefore, these organ weight changes were not interpreted to be toxicologically significant. There were no signs of neurotoxicity in the functional observational battery, motor activity test, reflex testing or grip strength. No inflammatory changes in kidneys were identified at any dose levels and the organ weight pattern including the kidney did not indicate an adverse effect at any dose level tested. The NOAEL for this study was 10000 ppm, the highest concentration tested in the diets (corresponding to 700-940 mg/kg bw/day).

In another experiment, 2 -phenoxyethanol was administered via gavage for 13 weeks to CD rats at doses of 0, 80, 400, and 2000 mg/kg bw/day (Nipa Laboratories, 1977). At 2000 mg/kg bw/day, clinical signs included occasional episodes of prostration and lethargy were observed shortly following 2-phenoxyethanol dosing. Females were more affected than males. These episodes occurred as isolated or low incidence events when dosing was initiated and resolved with continued dosing. Four high-dose females died during the treatment period and their deaths were considered treatment related, although no gross or histopathological changes were identified (one died of bronchopneumonia). The bodyweights of high-dose male and female rats were decreased. Clinical chemistry data showed toxicity to red blood cells and other effects that are associated with this phenomenon (decreased erythrocyte number, decreased packed cell volume and decreased haemoglobin concentration, and kidney inflammation with epithelial cells and polymorphonuclear leukocytes in the urinary sediment) at 2000 mg/kg bw/day. Liver, kidney and thyroid weights were increased at 2000 mg/kg bw/day. Inflammation of the kidneys also was seen in males at 400 mg/kg bw/day. Minor testicular changes were noted in a few high-dose male rats, but these changes were considered to be of equivocal toxicological significance. Furthermore, testicular changes have not been noted in any other studies with 2-phenoxyethanol. The NOAEL in this study was 80 mg/kg bw/day. The NIPA study, 90 day oral (gavage)study in rats is a pre-GLP study from 1977 n which dose levels of 80,400 and 2000 mg/kg bw were used. The highest dose level in this study by far exceeds the limit dose requested for repeated dose toxicity studies according to actual OECD guidelines (1000 mg/kg body weight). Moreover, the purity of the phenoxyethanol used in this study is unknown: it may have contained a high level of phenol. Due to the bolus dosing characteristic oral gavage is not the most relevant route of exposure in the view of the use of 2 -phenoxyethanol.

Comparing the two 90-day oral studies, it is possible that the difference in dosing regime (gavage versus diet) could account for the difference in toxicity observed between the studies. In the dietary study there were no overt signs of adverse effects at any dose level up to and including the highest dose of between 700 and 940 mg/kg bw/day. Conversely, in the gavage study evidence of kidney toxicity was observed at the mid dose of 400 mg/kg bw/day. One of the characteristics of EPh excretion is that there is active excretion and uptake of the main metabolite (PAA) the nephron which potentially concentrates the metabolite in the kidney. It is possible that following a gavage dose, higher levels of PAA are attained in the kidney compared to a similar or higher dietary dose. This may account for differences in the toxicity observed. However it should also be recognised that in the older gavage study, the purity of the EPh is unknown: it may have contained a high level of phenol.

For the purposes of hazard characterisation, gavage studies are considered to be less relevant than the dietary exposure due to the vast difference between how a human may be exposed and the typical bolus dose toxicity observed in gavage studies.

In addition to these studies, there are several shorter term studies in rats that demonstrate similar effects at similar doses. There are also comparative data in rats and rabbits that exemplify the species differences in sensitivity to hemolysis. It is clear from these two studies (Dow 1986 a and b) that rabbits are more sensitive to hemolysis than rats. Additional studies have since been performed to demonstrate that the toxicant is the parent compound, EPh, and that the basis for differences in sensitivity is the metabolic conversion to the acid metabolite. Both humans and rats metabolise EPh to PAA more rapidly than rabbits (refer to the toxicokinetics section). As such, repeated dose data generated using rabbits should be treated cautiously when assessing the repeated dose toxicity of EPh.

There are two oral, repeated dose toxicity studies (28d and 90d) performed by Unilever. Both studies dosed EPh in the diet to Wistar rats. Unfortunately only summaries of the data are available and as such it is challenging to make a full assessment of the data. From the limited information reported, the findings of these studies are generally consistent with those of the other oral studies. However the NOEL from the 90-d study is lower, 200 mg/kg bw, based on reduced body weight gain and evidence of liver induction in the high dose males (500 mg/kg bw/day). No adverse findings were reported for the females. Due to the very brief reporting of the data and the existence of a more recent GLP guideline 90-day study, the Unilever studies are given minimal weighting in the overall assessment of toxicity.

Chronic repeated dose studies:

Two repeated dose (104 weeks) OECD 451 and GLP compliant studies are available in rats and mice (JBRC.0498 and JBRC.0.497, 2007) (see also robust study summaries in IUCLID chapter 7.7). A drinking water study was conducted with F344/DuCrlCrlj rats. 50 rats per sex were exposed to nominal concentrations of 0, 2500, 5000, and 10000 mg/L. Analytical concentrations in drinking water were determined with HPLC. Based on chemical intake data the mean intake of test substance across the duration of the study was estimated to be 124, 249, and 510 mg/kg/day in males and 191, 380, and 795 mg/kg/day in females. Mortality and clinical signs were investigated. Food intake, water intake and body weight were determined weekly during the first 13 weeks followed by measurements once every 4 weeks until study termination. After 104 weeks urinalysis, haematology, blood chemistry, gross pathology at necropsy, organ weights and histopathology (both non-neoplastic and neoplastic lesions) were examined. No treatment related neoplastic lesions were found in either sex.

 

Another chronic drinking water study in B6 D2F1/Crlj mice was conducted. The study design and examination/observations were similar to the chronic study in rats. However, the dose levels differed and were 0, 5000, 10000 and 20000 mg/L. Based on chemical intake data the mean intake of test substance across the duration of the study was estimated to be 468, 898, and 1701 mg/kg/day for males and 586, 1072, and 2058 mg/kg/day for females. After 104 weeks repeated dosing, no treatment related neoplastic lesions were found in either sex.

 

Both chronic studies showed that there is no evidence of carcinogenic activity of 2-phenoxyethanol in male or female rat and mice. Although no carcinogenic effects were identified, effects on the blood chemistry parameters and histopathological changes were found that are in line with the above mentioned sub-chronic drinking water studies (JBRC 459 and 460, 2003). In the chronic drinking water study in mice cholesterol, phospholipids, and triglycerides were decreased in male mice and body weight was decreased in both sexes. Based on these results the NOAEL for mice was determined to be 468 mg/kg/day. In the chronic drinking water study in rats increased incidences and severity of renal pelvis urothelial hyperplasia were observed in male rats. Additionally, increased incidences of renal papillary mineralization and necrosis were observed in male rat. These non-neoplastic histopathological changes were minimal (grade 1 or 2) and were not observed in females. It was concluded that the kidney was the target organ in male but not female rats. Based on these results the NOAEL in the chronic drinking water study in rats was determined to be 249 mg/kg/day.

NOAEL/BMDL10 determination:

Several repeated dose studies are available were 2-phenoxyethanol was administered via the oral route. Among these studies a subchronic study involving dosing via diet (Bayer, 2002) and an additional study involving dosing by oral gavage are available, but the latter one was concluded of limited relevance/validity (Nipa Labs, 1977). In the Bayer study no adverse effects were detected up to the highest administered dose, approximately 700 mg/kg bw/d via diet.

In contrast several drinking water studies are available were effects were identified which could be used for derivation of a BMDL10. Histopathological findings from the available chronic as well as the sub-chronic drinking water studies indicate that the kidneys are the relevant target organs. Although treatment-related haemolytic effects were found in the sub-chronic drinking water studies in mice and rats, these effects occurred at higher dose levels compared to the effects on the kidneys. Therefore, the pathological findings in the kidneys were used to determine the NOAEL in the repeated dose drinking water studies. The urothelial hyperplasia of the renal pelvis in male rats was the most sensitive endpoint (JBRC.0459, 2003; JBRC 0497, 2007). Based on this effect the NOAELs were determined to be 248 and 369 mg/kg bw/d for male rats in the chronic and sub-chronic studies, respectively. The LOAELs were 510 and 687 mg/kg bw/d for male rats in the chronic and sub-chronic studies, respectively. Studies in mice showed higher NOAELs, thus, the studies with rats were considered as appropriately conservative for risk assessment.

Due to the fact that dose selections were different in the chronic and sub-chronic studies and that these dose selections influenced the NOAELs identified in these studies, a benchmark dose approach was used to determine a study-independent, “true” NOAEL, i. e. a BMDL10. The BMDL10 derived from the single studies were 356 and 350 mg/kg bw/d for the chronic and subchronic study, respectively. In addition, to get a more robust analysis, data from the chronic and sub-chronic studies in male rats were combined and the combined BMDL10 was determined to be 369 mg/kg bw/d. Combining both studies was justified, because progression of the renal effects was not observed with increased exposure duration. The BMDL10 of 369 mg/kg bw/d corresponds to the NOAEL from the subchronic drinking water study and lies between the LOAEL of 510 mg/kg bw/d and the NOAEL of 248 mg/kg bw/d of the chronic study. In conclusion, a BMDL10 of 369 mg/kg bw/d was considered as an appropriately conservative point of departure for DNEL-derivaton (DNEL(oral, long-term)).

Additional repeated dose oral toxicity studies:

Two 14 -days dose range finding studies were conducted in rats and mice (JBRC.0453 and JBRC.0454, 2003). In a drinking water study F344/DuCrj rats and DBF1 mice were exposed to 0, 1600, 4000, 7000, 10000, 17500, and 25000 mg/L (nominal) test substance. Clinical signs, mortality, food consumption, water consumption and body weight were observed or measured on the 3rd and 7th study day. After 14 days of exposure haematology, clinical chemistry and urinalysis were measured. Finally the rats were sacrificed and gross and histopathology were conducted. In rats, no mortality was observed in both sexes. Body weight growth, food intake and water intake were suppressed in both sexes in the higher dose groups. Significant changes in haematology were observed in all dose groups but predominantly in the higher dose groups (≥ 17500 mg/L). Significant effects on clinical chemistry were observed in rats dosed with ≥ 17500 mg/L test substance. Relative organ weights (liver and kidneys) were increased in the higher dose groups (≥10000 mg/L). In mice, no mortality was observed in both sexes. In the 25000 mg/L dose group body weight growth was suppressed in both sexes. Food intake was suppressed in the higher dose groups as well as water intake (≥10000 mg/L). No significant changes in haematology were observed. In female mice increased urea nitrogen concentration were measured after administration of 25000 mg/L test substance. No changes in clinical chemistry were observed in male mice. Increase in kidney weights were observed in the higher dose groups (≥7000 mg/L). These results were used to determine the dosage of a 13-week drinking water study in rat and mice described above.

Dermal:

In a 90-day repeated-dose dermal toxicity study, white rabbits were administered 0, 50, 150, and 500 mg/kg bw/day 2-phenoxyethanol for 6 h/day for 5 days/week during 13 weeks (The Dow Chemical Company, 1986; Breslin et al. 1991). There were no signs of systemic toxicity. Dermal findings included transient observations of erythema and slight scaling in rabbits exposed to 500 mg/kg bw/day. There were no associated gross or histopathological findings, thus, these dermal observations were not interpreted to be toxicologically significant. Therefore, 500 mg/kg bw/day was considered to be the NOAEL for systemic toxicity.

In addition to the 90-day study, 2 shorter term rabbit studies (14 days) are available. In the first (Dow 1985), a single dose level of 1000 mg/kg bw was applied once daily under occlusive conditions to 10 female rabbits. Three animals died or were euthanized, all showing signs of hemolysis, the remaining seven animals there were minimal findings relative to control, and in particular there was no evidence of hemolysis. In the second study (Hoescht Celanese, 1993) the test material was a 4% aqueous solution and as such the dose applied, 2000 mg/kg bw, was actually only 80 mg/kg bw of active. There were no adverse effects noted.

As indicated above, one of the key toxic effects of EPH in rabbits is hemolysis. Given their greater sensitivity to this effect relative to humans, the repeated dose toxicity data generated using rabbits should be treated cautiously. Therefore the NOEL of 500 mg/kg bw/day from the 90-day dermal study in rabbits represents a conservative assessment of the repeated dose dermal toxicity of EPh.

Inhalation:

The inhalation study was performed to characterize the toxicity profile of 2-phenoxyethanol including target organs and determination of a NOAEL upon exposure to a liquid aerosol (BASF AG, 2007). Special emphasis was paid on potential irritation in the respiratory tract. No mortality occurred and no signs of systemic toxicity were observed. There were no treatment-related changes in the clinical-chemical and haematological parameters. Body weight was reduced in the high dose group. The upper respiratory tract showed mild signs of reaction (degeneration, metaplasia, inflammatory cell infiltration); most sensitive seemed to be the respiratory epithelium of the nose, especially in the anterior septum area. The epithelial lining other sites, e. g. respiratory epithelium of lungs, transitional epithelium larynx was also affected but appeared to be less sensitive. The base of the epiglottis was covered by metaplastic squamous epithelium in high concentration males and females and one female of the mid-dose group. Pathological examinations revealed no treatment-related changes in either males or females. Morphological changes indicating irritation were found in nasal cavity, larynx and lung of male and female mid- and high-concentration animals. A NOAEC of 48.2mg/m³ was determined.

Based on the use of read across to data from EPh, the following molecular weight corrected NOELs will be taken forward to the hazard and risk characterization:

Oral: 489.5 mg/kg bw/day (used for oral and inhalation systemic DNELs)

Dermal: 659.5 mg/kg bw/day (used for dermal systemic DNEL)

Inhalation: 63.6 mg/m3 (used for local effects assessment only)

The oral BMDL10 based on combining the subchronic and chronic repeated dose toxicity studies will be used to determine the oral and inhalation DNELs. The NOEL from the 90 -day rabbit study will be used to derive the dermal DNEL.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
most reliable study for the endpoint - data taken from the analogue substance

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
only study available to assess local effects following inhalation - data from analogue.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Most reliable 90-day repeated dose toxicity study- data from analogue.

Justification for classification or non-classification

The available data for Di-EPh and similar material EPh are conclusive but not sufficient for classification.

Oral: no classification (effects above the cut-off values)

Dermal: no classification (effects above the cut-off values)

Inhalation: no classification (no relevant effects in the lung: irritation potential in the upper respiratory tract)