Registration Dossier

Administrative data

Description of key information

28 day repeat dose oral study in rat: NOAEL 50 mg/kg/day.
90 day repeat dose dermal study in rabbit: NOAEL 500 mg/kg/day (Read-across from 2-phenoxyethanol)

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:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant with current guidelines and GLP compliant
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 62 days of age (males) and 64 days of age (females)
- Weight at study initiation: The body weight range for the male rats was 228 g to 265 g on the day after arrival at the Testing Facility and was 256 g to 290 g at randomization. The body weight range for the female rats was 172 g to 214 g on the day after arrival at the Testing Facility and was 189 g to 210 g at randomization.
- Housing: The rats were individually housed in stainless steel, wire-bottomed cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days (males) and 8 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 30% to 70%
- Air changes (per hr): A minimum of 10 changes per hour of 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hours light: 12-hours dark fluorescent light cycle

IN-LIFE DATES: From: To: 07 Nov 2011 to 19 Dec 2011
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Commonly used vehicle with test material not soluble in water.
- Concentration in vehicle: 10, 30 and 90 mg/mL
- Amount of vehicle (if gavage): 5 mL/day
- Lot/batch no. (if required): M-631
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing suspensions of the test substance prepared in corn oil were analyzed and were found to be acceptable and homogeneous under the conditions of the study. At the initiation of dose administration, all prepared formulations used for dose administration were analyzed and were found to be -0.7%, 0.7% and 1.6% from the target concentrations of 10 mg/mL, 30 mg/mL and 90 mg/mL, respectively. The corresponding percentage differences for the analyses conducted at the end of the study were -0.2%, -3.3% and -5.3%, respectively. The homogeneity values obtained were +0.7%, +1.2% and +2.2% RSD for the 10 mg/mL, 30 mg/mL and 90 mg/mL formulations, respectively.
The stability of the prepared test substance formulations bracketing the range of concentrations used on study was determined in Charles River Laboratories study number 20016972. Stability was determined in the 1 mg/mL and 200 mg/mL dosing solutions at room temperature (20 °C to 25 °C) for 25 hours and refrigerated (2 °C to 8 °C) for 10 days.
Duration of treatment / exposure:
28 days
Frequency of treatment:
The rats received the test substance for 28 consecutive days.
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
450 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 rats/sex/dose group; 5 rats/sex/dose group in the 0 and 450 doses for the satellite group
Details on study design:
- Dose selection rationale: Based on a 14 day range finder (detailed as a supporting study IUCLID 7.5.1).
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Random
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): Random
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The rats were observed for general appearance once during the acclimation period, daily prior to dose administration and daily during the postdose period.
- There were 29 observations performed on the main study animals and 43 observations performed on the recovery animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were recorded once prior to initiation of dose administration and weekly thereafter (with the exception of those weeks in which the Functional Observational Battery (FOB) was conducted since detailed clinical observations were subsumed within the FOB examination).

BODY WEIGHT: Yes
- Time schedule for examinations: Daily prior to dose administration

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

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Collected on the day of euthanasia from the inferior vena cava.
- Anaesthetic used for blood collection: Yes Isoflurane/oxygen was used as the anaesthetic.
- Animals fasted: Yes They were fasted for no longer than 24 hours.
- How many animals: All animals in the main study and the recovery portion were evaluated.
- Parameters assessed: Red blood cell count, Hemoglobin concentration, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Reticulocyte count (absolute), Mean platelet volume, Platelet count, White blood cell count, Neutrophil count, Lymphocyte count, Monocyte count, Eosinophil count, Basophil count, Large unstained cells


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Collected on the day of euthanasia from the inferior vena cava.
- Animals fasted: Yes They were fasted for no longer than 24 hours.
- How many animals: All animals in the main study and the recovery portion as well.
- Parameters assessed: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Total bilirubin, Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride


URINALYSIS: Yes
- Time schedule for collection of urine: Day 29 of exposure (main study) and day 43 of study (recovery portion)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters assessed: Colour, Clarity, Specific gravity, Microscopic evaluation of urine sediment, Total Volume, pH, Protein, Glucose, Bilirubin, Ketones, Nitrites, Leukocytes, Blood, Urobilinogen


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional observational battery and motor activity were evaluated on day 28 of the study.
- Dose groups that were examined: All dose groups were examined.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: There were also evaluations of sensorimotor responses to visual, acoustic, tactile and painful stimuli (reactivity and sensitivity) as well as air righting reaction, visual placing response, landing foot splay and body temperature.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Test items in the FOB using interval scales, such as the grip-strength tests and the landing foot splay test, as well as body weight data and feed consumption values were analyzed as described under the Parametric heading of the schematic. Bartlett's Test of Homogeneity of Variances was used to estimate the probability that the groups had different variances. A nonsignificant result (p> 0.001) indicated that an assumption of homogeneity of variance was not inappropriate, and the data was compared using the Analysis of Variance Test. If that test was significant (p≤ 0.05), the groups exposed to the test substance were compared with the control group using Dunnett's Test If Bartlett's Test was significant (p≤ 0.001), the Analysis of Variance Test was not appropriate, and the data were analyzed as described under the Nonparametric heading of the schematic. When 75% or fewer of the scores in all the groups were tied, the Kruskal-Wallis Test was used to analyze the data, and in the event of a significant result (p≤ 0.05), Dunn's Test was used to compare the groups exposed to the test substance with the control group. When more than 75% of the scores in any group were tied, Fisher's Exact Test was used to compare the proportion of ties in the groups.

The remaining information regarding the statistical analyses are provided in the following section.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Male and female rats at 450 mg/kg/day
Mortality:
mortality observed, treatment-related
Description (incidence):
Male and female rats at 450 mg/kg/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduction in body weights and body weight gains in the male rats at 450 mg/kg/day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduction in absolute and relative food consumption in male rats at 450 mg/kg/day
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Male and female rats at 150 and 450 mg/kg/day
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Increase in movement in female rats at 450 mg/kg/day at the end of the dose period.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY

No test substance-related deaths occurred during the course of the study.
One male rat in the 450 mg/kg/day dose group was found dead on day 10 of study (DS 10). Adverse clinical signs noted for this rat included low carriage (DSs 1 to 3), decreased motor activity (DS 2), mild dehydration (DS 4) and ungroomed coat (DS 8). This rat had comparable body weight and feed consumption values in comparison with other rats in this dose group. Necropsy revealed that all lobes of the lungs were mottled red and dark red; all other tissues evaluated appeared normal. Based on the necropsy observation, this death was considered to be the result of an intubation error and not test substance-related.

There was an increase or statistically significant increase in the number of male and female rats in the 450 mg/kg/day dose groups observed with excess salivation (slight, moderate or extreme) and low carriage in comparison with the control group value. There was also an increase in the number of male and female rats observed with mild or moderate dehydration and the number of male rats observed with urine-stained abdominal fur.

BODY WEIGHT AND WEIGHT GAIN

In the male rats at 450 mg/kg/day, there were statistically significantly reduced (p≤ 0.05 to p≤ 0.01) mean body weights and body weight gains observed at all intervals during the dose period (DSs 1 to 28). In the male rats, body weight gains for the overall dose period (DSs 1 to 28) in the 50, 150 and 450 mg/kg/day dose groups were 86.9%, 102.9% and 61.9%, respectively, as compared with the control group value. The body weight gain for the male rats in the 450 mg/kg/day dose group during the recovery period (DSs 29 to 42) was 89.6% as compared with the control group value.
In the female rats, body weight gains for the overall dose period in the 50, 150 and 450 mg/kg/day dose groups were 112.0%, 81.7% and 98.6%, respectively, as compared with the control group value. The body weight gain for the female rats in the 450 mg/kg/day dose group during the recovery period was 170.7% as compared with the control group value.

HAEMATOLOGY
At the end of the dose period, there was a statistically significant increase (p≤0.05) in the mean platelet volume observed in the female rats at 450 mg/kg/day in comparison with the control group value. In the female rats at 450 mg/kg/day, there was also a statistically significant decrease (p≤0.05) in the mean corpuscular hemoglobin concentration in comparison with the control group value. These differences were not considered to be test substance-related because the values were within the historical control range of the Test Site performing the analysis.

At the end of the recovery period (DS 43), there was a statistically significant decrease (p≤0.05) in hemoglobin observed in the male rats at 450 mg/kg/day in comparison with the control group value. This difference was not considered to be test substance-related because the value was within the 95% spread of the Test Site historical control range.

There were no additional statistically significant or marginally different hematologic changes observed in the male or female rats in this study.

CLINICAL CHEMISTRY

At the end of the dose period (DS 29), changes in serum chemistry that were presumed related to treatment with the test substance included:

• a statistically significant decrease (p≤0.05) in aspartate aminotransferase was observed in the male and female rats at 150 and 450 mg/kg/day;
• an increase or statistically significant increase (p≤0.05) in triglyceride was observed in the male and female rats at 150 and 450 mg/kg/day;
• a statistically significant increase (p≤0.05) in glucose was observed in the male and female rats at 450 mg/kg/day; and
• a statistically significant increase (p≤0.05) in the albumin/globulin ratio and the total bilirubin was observed in the male rats at 450 mg/kg/day.

By the end of the recovery period (DS 43), these changes in serum chemistry had resolved.

All other changes in serum chemistry were not attributed to treatment with the test substance. There was a statistically significant increase (p≤0.05) in cholesterol observed in the female rats at 150 and 450 mg/kg/day in comparison with the control group value. There was also a statistically significant increase (p≤0.05) in creatinine observed in the male rats and calcium observed in female rats at 450 mg/kg/day in comparison with the control group value. These differences were not considered to be test substance-related because the values were within the 95% spread of the Test Site historical control range.

URINALYSIS

There were no biologically important differences between the control and treated groups in the urinalysis parameters evaluated on DSs 29 or 43. In the male rats on DS 29, the specific gravity of the urine in the 50, 150 and 450 mg/kg/day dose groups was significantly lower (p≤ 0.05 to p≤ 0.01) than the control group value (range of 1.0128 to 1.0180 vs. 1.0255 in control group). These differences were not considered to be test substance-related because the control group value appears to be high in comparison with the Testing Facility historical control range (range of 0.8042 to 1.0220).

NEUROBEHAVIOUR

Exposure to the test substance at doses as high as 450 mg/kg/day to male and female rats did not affect any of the parameters evaluated in the functional observational battery (FOB). There was a statistically significant increase (p≤0.05) in the total number of movements observed during the motor activity evaluation performed at the end of the dose period for the female rats at 450 mg/kg/day.

ORGAN WEIGHTS

At the completion of the dose period (DS 29), there was a statistically significant reduction (p≤0.01) in terminal body weights in the male rats at 450 mg/kg/day in comparison to the control group value. Terminal body weights in the male rats at 450 mg/kg/day remained reduced relative to the control group value during the recovery period. Organ weights (absolute and relative) were unaffected by up to 450 mg/kg/day of the test substance. All other statistically significant findings were considered unrelated to the test substance because: 1) the changes were not dosage-dependent; and/or 2) the observation did not persist during the recovery period; and/or 3) the observation was associated with the reduction in body weight.

GROSS PATHOLOGY

There were no test substance-related necropsy observations apparent at the completion of the dose period as well as the recovery period. All necropsy observations were considered unrelated to the test substance because the observations occurred in single rats in the dose groups.

HISTOPATHOLOGY: NON-NEOPLASTIC

No test substance-related microscopic findings were noted on study. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals.

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At the end of the dose period, changes in serum chemistry that were considered to be test substance-related included a decrease in aspartate aminotransferase and an increase in triglyceride in the male and female rats at 150 and 450 mg/kg/day.
Critical effects observed:
not specified
Conclusions:
The no-observed-adverse-effect-level (NOAEL) for systemic toxicity produced by oral exposure to the test substance is considered to be at least 50 mg/kg/day for male and female rats. At 150 and 450 mg/kg/day, changes in clinical chemistry parameters were observed in both the male and female rats. In the 450 mg/kg/day dose group, there were also adverse clinical signs observed in both the male and female rats. In the male rats at 450 mg/kg/day, the body weights, body weight changes and food consumption values were reduced throughout the dose period.
Executive summary:

Executive Summary

 

The objectives of this study were to determine the potential toxicity of EC 202-228-8 (test substance), when given orally via gavage for 28 days to rats, to evaluate the potential reversibility of any findings.

The study design was as follows as detailed in Text Table1:

Text Table1
Experimental Design

Group No.

Number of Rats per Sex

Test Material

Dose Level

(mg/kg/day)

Concentration

(mg/mL)

Dose Volume

(mL/day)

Main Study

Recovery Portion

1

10

5

Corn Oil

0

0

5

2

10

-

Test Substance

50

10

5

3

10

-

Test Substance

150

30

5

4

10

5

Test Substance

450

90

5

- = Not applicable

Rats were administered the test substance and/or the vehicle, corn oil, once daily by oral gavage from Days 1 through 28 of study. The dose volume was 5 mL/kg and doses were based on the most recent body weights recorded prior to dose administration and given at approximately the same time each day.

The following parameters and end points were evaluated in this study: viability, clinical signs, body weights, body weight changes, food consumption, functional observational battery, motor activity, clinical pathology, gross necropsy findings, histology and histopathology.

On Day 29 of study (main study) or Day 43 of study (recovery portion), all surviving rats were anesthetized under isoflurane/oxygen and following blood collection from the inferior vena cava, were subsequently euthanized by an injection of sodium pentobarbital into the inferior vena cava. Examination for gross lesions, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. 

No deaths related to the test substance occurred. One male rat in the 450 mg/kg/day dose group was found dead on Day 10 of study (DS 10). Necropsy revealed that all lobes of the lungs were mottled red and dark red. Based on the necropsy observation, this death was considered to be the result of an intubation error. 

There was an increase or statistically significant increase in the number of male and female rats in the 450 mg/kg/day dose groups observed with excess salivation (slight, moderate or extreme) and low carriage. There was also an increase in the number of male and female rats observed with mild or moderate dehydration and the number of male rats observed with urine-stained abdominal fur. 

In the male rats at 450 mg/kg/day, there were statistically significantly reduced mean body weights and body weight gains observed at all intervals during the dose period. Absolute and relative food consumption values were also reduced or statistically significantly reduced in the male rats at 450 mg/kg/day at all intervals during the dose period. 

At the end of the dose period, changes in serum chemistry that were considered to be test substance-related included a decrease in aspartate aminotransferase and an increase in triglyceride in the male and female rats at 150 and 450 mg/kg/day. There was an increase in glucose observed in the male and female rats at 450 mg/kg/day. In the male rats at 450 mg/kg/day, there was also an increase in the albumin/globulin ratio and the total bilirubin observed at the end of the dose period. These changes in serum chemistry had resolved by the end of the recovery period. 

Doses of the test substance as high as 450 mg/kg/day for 28 consecutive days did not cause any findings during the FOB and motor activity evaluations on DS 28. Likewise, the urinalysis performed at the end of dose period for both male and female rats showed no adverse effects. There were no adverse gross lesions observed at necropsy, effects on the organ weights, hematology or gross or microscopic histopathologic changes that were attributed to exposure to the test substance.   

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity produced by oral exposure to the test substance is considered to be at least 50 mg/kg/day for male and female rats. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Modern GLP study conducted in accordance with OECD test guideline, Klimisch score 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Published May 3rd 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study comparable to guideline study, performed on a suitable analogue substance.
Qualifier:
no guideline followed
Principles of method if other than guideline:
No guideline stated in the publication. Study conducted equivalent or similar to OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study).
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazleton-Dutchland, Denver, PA.
- Age at study initiation: approx. 5 months
- Weight at study initiation: Not stated.
- Fasting period before study: Not stated.
- Housing: Individually
- Diet: ad libitum (Certified Laboratory Rabbit Chow, Ralston Purina Co., St. Louis, MO)
- Water: ad libitum
- Acclimation period: minumum of 3 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 22
- Humidity (%): 50%
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
EPGE was uniformly spread over clipped area (approximately 10x15 cm) using a syringe and blunt tipped needle. The application site was reclipped as required during the study to keep it free of hair. An occlusive wrap of absorbent gauze and non absorbant cotton was placed over the application area and was held in place using an elastic jacket. The wraps and jackets were removed approximately 6 hours after each dose was applied. The backs of the rabbits were not wiped since no appreciable amount of test material was apparent at the site of application after 6 hour exposure period.

Dosing volumes were approximately 0.05 to 0.50 ml/kg/day and was adjusted weekly based upon body weight.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week for thirteen 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: The top dose level of the study (500 mg/kg/day) was selected based on the results of previous studies, in which hemolytic effects/mortality was observed following 12 consecutive daily dermal doses of 600 mg/kg/day.

- Randomization of test animals to treatment groups was performed using a computer generated randomization scheme designed to produce groups with equivalent mean body weight and variance. All aminals were identified by means of a uniquely numbered metal ear tag.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes

All animals were observed daily throughout the test period for indications of toxicity. Animals found dead or moribund or surviving the treatment period were submitted for a complete gross pathological examination by a veterinary pathologist.

DERMAL IRRITATION (if dermal study): Yes

The condition of the skin at the site of test material application was assessed and recorded prior to dosing for the first two weeks and approximately weekly thereafter using the laboratory's modification of the draize system.

BODY WEIGHT: Yes

All rabbits were weighed prior to the first exposure and weekly thereafter.

FOOD CONSUMPTION: No data.


WATER CONSUMPTION: No data.


OPHTHALMOSCOPIC EXAMINATION: No data.


HAEMATOLOGY: Yes

Blood samples (approximately 7 mL) for hematology and clinical chemistry determinations were obtained from the auricular artery of all rabbits approximately 1 week prior to dosing and after 4 and 13 weeks of treatment. Samples for hematological determinations were collected using vacuum tubes and mixed with potassium EDTA anticoagulant. RBC, WBC, and PLAT counts, HGB concentration, PCV, and RBC indices (MCV, MCH, MCHC) were determined on all samples. Differntial leukocyte and reticulocyte (RETICS) counts and RBC morphological determinations were condicted on samples from all control and high dose rabbits by direct examination of stained smears after 13 weeks of treatment only.

CLINICAL CHEMISTRY: Yes

Blood samples for clinical chemistry determinations were allowed to clot at 0-5 degrees C and serum was harvested by centrifugation. Alanine aminotransferase activity, aspartate aminotransferase activity, glucose, total protein, albumin, globulin and total bilirubin were determined on all samples.

URINALYSIS: No data.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

On the day following the last dermal application of EGPE, each rabbit was euthanatized with CO2, weighed and examined for gross pathological alterations. Brain. heart, liver, kidneys, and testes were weighed and relative organ weights (g/l00 g body weight) were calculated.

HISTOPATHOLOGY: Yes

A representative sample of all organ systems and tissues were collected from each rabbit at necropsy and preserved in neutral, phosphate-buffered 10% formalin. All tissues collected from control and high dose animals were processed by conventional histologic techniques, stained with
haematoxylin and eosin and evaluated by light microscopy.
Statistics:
Body weight, organ weight and appropriate haematology and clinical chemistry data were evaluated by Bartlett’s test (α = 0.01) for the equality of variances. Based upon the outcome of Bartlett’s test, a parametric or nonparametric analysis of variance (ANOVA) was conducted followed by Dunnett’s test or the Wilcoxon rank-sum test with Bonferroni’s correction (α = 0.10) if the ANOVA was significant. The nominal level of significance used for comparing results obtained from control and treated groups was 0.05. Statistical outliers were identified by a sequential outlier test but were not excluded from analysis.
Clinical signs:
no effects observed
Description (incidence and severity):
No overt signs of systemic toxicity (including hemoglobinuria) or early mortality of treated rabbits were noted over the 13 week period.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
See Details on Results below.
Mortality:
no mortality observed
Description (incidence):
No overt signs of systemic toxicity (including hemoglobinuria) or early mortality of treated rabbits were noted over the 13 week period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment related effects upon body weight.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment related effects on haematologic parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment related effects on clinical chemistry parameters.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment related effects on organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross changes attributed to EGPE.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic changes attributed to EGPE.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No microscopic changes attributed to EGPE.
Details on results:
The only potentially treatment related effect was the sporadic observation of erythema and very slight-to-slight scaling of the skin at the site of test material application in male and female rabbits exposed to 500 mg EGPE/kg/day. However, as these effects were not associated with gross or histologic changes in the skin they were not considered to be of toxicologic significance.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of significant toxicological effects at all exposure levels.
Dose descriptor:
LOAEL
Effect level:
> 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of significant toxicological effects at all exposure levels.
Critical effects observed:
not specified
Conclusions:
Dermal administration of up to 500 mg/kg bw/day EGPE (the highest dose tested) for 90 days had no effect in rabbits other than sporadic observation of erythema and very slight to slight scaling of the skin at the test site. However as these effects were not associated with gross or histological changes in the skin they were not considered to be of toxicological significance. The NOAEL was therefore concluded to be 500 mg/kg bw/day.
Executive summary:

Dermal administration of up to 500 mg/kg bw/day (the highest dose tested) of ethylene glycol phenyl ether (2-phenoxyethanol) for 90 days had no effect in rabbits other than sporadic observation of erythema and very slight to slight scaling of the skin at the test site. As these effects were not associated with gross or histological changes in the skin they were not considered to be of toxicological significance.

In conclusion, no systemic toxicity was seen when rabbits were dermally exposed to 2-phenoxyethanol for 13 weeks at doses of up to 500 mg/kg bw/day, under conditions reflective of potential human occupational exposure.

The data presented for the analogue substance, 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 provided within IUCLID section 13.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rabbit
Quality of whole database:
Study conducted equivalent or similar to OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study).

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
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 signficant 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 out 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, there is low risk to human health, as such 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 for2-phenoxyethanol reflects the toxicological profile of 2-(2-naphthyloxy)ethanol, as such read-across between the two substances is acceptable. It is considered that sub chronic repeated dose 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-acros between 2-phenoxyethanol and 2-(2-naphthyloxyethanol) is attached to section 13 of this dataset.

Repeated dose toxicity - Oral

A modern GLP study conducted in accordance with the OECD 407 guideline was designed to evaluate the potential toxic effects of the Registration substance when administered via oral gavage to rats for 28 days.

For a period of twenty-eight consecutive days the Crl:CD(SD) strain rats were dosed with a EC 202-228-8 at dose levels of 50, 150 and 450 mg/kg/day.

No deaths related to the test substance occurred. One male rat in the 450 mg/kg/day dose group was found dead on Day 10 of study (DS 10). Necropsy revealed that all lobes of the lungs were mottled red and dark red. Based on the necropsy observation, this death was considered to be the result of an intubation error. 

There was an increase or statistically significant increase in the number of male and female rats in the 450 mg/kg/day dose groups observed with excess salivation (slight, moderate or extreme) and low carriage. There was also an increase in the number of male and female rats observed with mild or moderate dehydration and the number of male rats observed with urine-stained abdominal fur. 

In the male rats at 450 mg/kg/day, there were statistically significantly reduced mean body weights and body weight gains observed at all intervals during the dose period. Absolute and relative food consumption values were also reduced or statistically significantly reduced in the male rats at 450 mg/kg/day at all intervals during the dose period. 

At the end of the dose period, changes in serum chemistry that were considered to be test substance-related included a decrease in aspartate aminotransferase and an increase in triglyceride in the male and female rats at 150 and 450 mg/kg/day. There was an increase in glucose observed in the male and female rats at 450 mg/kg/day. In the male rats at 450 mg/kg/day, there was also an increase in the albumin/globulin ratio and the total bilirubin observed at the end of the dose period. These changes in serum chemistry had resolved by the end of the recovery period. 

Doses of the test substance as high as 450 mg/kg/day for 28 consecutive days did not cause any findings during the FOB and motor activity evaluations on DS 28. Likewise, the urinalysis performed at the end of dose period for both male and female rats showed no adverse effects. There were no adverse gross lesions observed at necropsy, effects on the organ weights, hematology or gross or microscopic histopathologic changes that were attributed to exposure to the test substance.   

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity produced by oral exposure to the test substance is considered to be at least 50 mg/kg/day for male and female rats. 

The doses selected for the 28 study were based on a non-GLP 14 day rangefinder. The high-dose group animals were initially dosed at 1000 mg/kg/day and showed increased incidences of mortality, adverse clinical signs, body weight loss and decreased food consumption. Therefore the dose for this group was reduced to 600 mg/kg/day on day 9 of the study after which the adverse effects persisted. Although all five of the male rats assigned to the main study assigned to the 1000 mg/kg/day dose level were either found dead or were euthanized based on their adverse clinical signs and body weight loss, it is possibly this was a carry-over from the initial dosing at 1000 mg/kg/day. The three females that remained on study experienced a body weight gain as opposed to a body weight loss and an increase in feed consumption values. Therefore, male rats were more sensitive to this test substance and additional rats were evaluated in order to provide the necessary information to assist in establishing doses for the 28-day study. The study was extended to evaluate 5 additional male rats at the 600 mg/kg/day dose level for 7 consecutive days. The extension study established that the 600 mg/kg/day dosage alone would not be well tolerated as the same adverse clinical signs, body weight loss and decreased food consumption were observed after a 7-day dosage period.

In the rats at 300 mg/kg/day, there was an initial body weight loss observed in the female rats and a decrease in food consumption observed in both sexes on the first day of dosage administration. However, these effects quickly rebounded, and this dose was well tolerated for the remainder of the study.

Repeated dose toxicity - dermal

No dermal repeated dose toxicity data is available for the registration substance, however, there is a 90 day repeated dose toxicity study available for 2-phenoxyethanol (EC 204-589-7). It is considered that read-across between the registration substance and 2-phenoxyethanol is appropriate due to the structural similarity of the two substances. This structurally similarity will dictate that toxicokinetic and toxicological profiles will be similar. Therefore the 90 day repeated dose toxicity study for 2-phenoxyethanol will be used as read-across to 2 -(2 -naphthyloxyethanol), this will act as the sub-chronic study for the registration substance on which DNEL derivation and classification and labelling will be based. The justification for the use of this read across is located within IUCLID section 13.

Dermal administration of up to 500 mg/kg/day 2-phenoxyethanol (the highest dose tested) for 90 days had no effect in rabbits other than sporadic observation of erythema and very slight to slight scaling of the skin at the test site. However as these effects were not associated with gross or histological changes in the skin they were not considered to be of toxicological significance.

The difference in response observed between this dermal study and the dermal teratology study (Scortichini et al 1987, presented in IUCLID section 7.8.2) is likely due to differences in the absorption of the 2-phenoxyethanol. In the dermal teratalogy study, the continuous exposure site occlusion method used may have contributed to alterations in the integrity of the epidermis as indicated by skin lesions in affected animals, resulting in enhanced absorption of the test material and subsequent development of hemolytic anemia.

The continuous 24 hour occlusion and repeated dosing under the wrap most likely prevented adequate air flow to the dermal exposure site increasing moisture retention and hydration of the stratum corneum. Increased hydration of the stratum corneum has previously been shown to result in a 2X increase in the permeability constant for various alcohols (Blank, 1985).

In the 90-day dermal repeat dose study, an attempt was made to more closely reflect the potential intermittent exposure of 2-phenoxyethanol during manufacture and use by restricting 2-phenoxyethanol treatment to 6 hr/day, 5 days/week and changing occlusive wraps daily. In addition, in this study the wraps were held in place using an elastic open-weave jacket designed to allow adequate air flow to the skin, preventing excessive moisture retention and dermal hydration.

In conclusion, dermal exposure of rabbits to high doses (500 mg/kg/day) of 2-phenoxyethanol under conditions reflective of potential human exposure, does not result in systemic toxicity.

Repeated dose toxicity - inhalation

In accordance with column 2 of Section 8.6 of REACH Annexes VIII and IX, it is considered justifiable to omit repeated dose toxicity testing via inhalation. The registration substance is a solid and it will not be aerosolized in its normal use pattern and is not anticipated to form small particles or droplets. As such testing via the inhalation route is not considered appropriate for this substance.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A key study for repeat dose toxicity, oral exposure was conducted in accordance with OECD Guideline 407(28-day repeat dose study) and GLP. Klimisch score 1.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
In accordance with Section 8.6.1 of Annex VIII of the REACH Regulation, a study of the subacute (28-day) inhalation toxicity of the test substance is not required, since a study of the subacute (28- day) toxicity of the Registration substance via the oral route is provided (see IUCLID dossier Section 7.5.1). This is considered a more relevant route as there is very low potential for inhalation exposure to the substance.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
In accordance with Section 8.6.1 of Annex VIII of the REACH Regulation, a study of the subacute (28-day) inhalation toxicity of the test substance is not required, since a study of the subacute (28-day) toxicity of the Registration substance via the oral route is provided (see IUCLID dossier Section 7.5.1). This is considered a more relevant route as there is very low potential for inhalation exposure to the substance.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The 90 day repeat dose dermal study is selected as the key effect level, this is based on the attempts to more closely reflect the potential intermittent exposure during manufacture and use by restricting treatment to 6 hr/day, 5 days/week and changing occlusive wraps daily.

This study is chosen in preference to the dermal developmental toxicity/teratogenicity study (IUCLID section 7.8.2) in which significant toxicity was observed. This study involved 24 hour occlusive dermal exposure, the continuous exposure site occlusion method used may have contributed to alterations in the integrity of the epidermis as indicated by skin lesions in affected animals, resulting in enhanced absorption of the test material and subsequent development of hemolytic anemia.

Therefore the 90 day repeat dose study is considered more representative of potential exposure and is the more appropriate NOAEL to select as the key effect level.

The data presented for material 2-phenoxyethanol (EC 204-589-7) is considered appropriate for read-across to EC 202-228-8 and to aid 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 repeated dose toxicity dermal - local effects endpoint:
The Registration substance is not a skin irritant or a skin sensitiser - see IUCLID Sections 7.3 and 7.4.

Justification for classification or non-classification

A 28-day oral toxicity study in rats identified a NOAEL of 50 mg/kg bw/day. A weight of evidence approach was undertaken to conclude on classification of EC-202-228-8 as outlined in CLP. It was concluded that EC-202-228-8 does not require a STOT classification for repeated exposure under CLP and the justification for this decision is detailed below:

The non-GLP 14 day rangefinder demonstrated that high doses of EC 202-228-8 (1000/600 mg/kg bw/day) were not well tolerated, with increased incidences of mortality, adverse clinical signs, body weight loss and decreased food consumption. This dictated dose level selection for the 28 day study.

In the 28 day study there was a statistically significant increase in the number of male and female rats in the 450 mg/kg/day dose groups observed with excess salivation (slight, moderate or extreme) and low carriage. There was also an increase in the number of male and female rats observed with mild or moderate dehydration and the number of male rats observed with urine-stained abdominal fur. In the male rats at 450 mg/kg/day, there were statistically significantly reduced mean body weights and body weight gains observed at all intervals during the dose period. Absolute and relative food consumption values were also reduced or statistically significantly reduced in the male rats at 450 mg/kg/day at all intervals during the dose period. It was considered that these effects were likely due palatability of the test material, did not indicate significant toxicity, and were not of the nature of effect that might require classification for specific target organ toxicity (STOT), furthermore these effects were only seen at 450 mg/kg/day, which is above the guidance values outlined in CLP for STOT classification.

 

At the end of the dose period, changes in serum chemistry that were considered to be test substance-related included an increase in glucose observed in the male and female rats at 450 mg/kg/day. In the male rats at 450 mg/kg/day, there was also an increase in the albumin/globulin ratio and the total bilirubin observed at the end of the dose period. All of these changes were fully reversible at the end of the recovery period and could not be attributed to any organ toxicity. It was considered that these effects did not show evidence of significant toxicity or STOT and furthermore the effects occurred only above the guidance values set out in CLP STOT classification.

 

Other changes in serum chemistry included a decrease in aspartate aminotransferase and an increase in triglyceride in the male and female rats at 150 and 450 mg/kg/day. These changes in serum chemistry could not be attributed to toxicity in a specific organ as there were no adverse gross lesions observed at necropsy, no effects on the organ weights, no haematology or gross or microscopic histopathological changes associated with the test substance. The changes had also resolved by the end of the recovery period. All of these findings were thought to relate to adaptive metabolic changes, rather than indicative of particular organ toxicity. Although these effects were likely to be caused by the test material, they were not considered to be indicative of any significant toxicity. It is therefore concluded that EC 202-228-8 does not require a STOT classification for repeated exposure.

Dermal administration of up to 500 mg/kg/day 2-phenoxyethanol (the highest dose tested) for 90 days had no effect in rabbits other than sporadic observation of erythema and very slight to slight scaling of the skin at the test site. However as these effects were not associated with gross or histological changes in the skin they were not considered to be of toxicological significance. In conclusion, dermal exposure of rabbits to high doses (500 mg/kg/day) of 2-phenoxyethanol under conditions reflective of potential human exposure, does not result in systemic toxicity.

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.

 

The substance has not been tested for repeat-dose toxicity via inhalation exposure.