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Description of key information

The only repeat dose study available is a 90 day dermal study using a 57.58% solution of hydroxyethyl urea in water (EXP3982).  As dermal exposure is the most likely route of exposure, with very little possibility of inhalation or oral exposure, this study will be used as the basis for calculation DNELs.  Additional testing by the oral and inhalation routes is not considered to be scientifically justified. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
We have a Klimisch 1, 90 day dermal toxicity study which can be used to read across to the oral route of exposure. There is no measured data on dermal penetration but the model data does indicate dermal absorption can be expected to be the same as oral absorption so extrapolation to the oral route is acceptable.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
The requirement for a study is waived due to the availability of Klimisch 1 90 day study by the dermal route and the lack of potential for inhalation exposure to the product. The dermal NOEL will be used as a NOAEL with the additional assessment factor of 2 to provide an adequate systemic DNEL

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
There is a Klimisch 1 acute inhalation study on another product containing ca. 50% of hydroxyethyl urea. This did not show any specific local effects at the maximum dose level tested on 4mg/l. This is considered sufficient; it is not scientifically justified to perform additional animal tests to establish a NOAEL for local effects in the respiratory tract.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2001-04-18 to 2002-03-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline conform study
Qualifier:
according to
Guideline:
EPA OPPTS 870.3250 (Subchronic Dermal Toxicity 90 Days)
Deviations:
yes
Remarks:
: eight listed deviations which according to the author did not affect integrity or validity of the study from a scientific or GLP perspective.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc, Portage, Michigan
- Age at study initiation: approximately nine weeks of age
- Weight at study initiation: 258-305 g for males and 193-227 g for females
- Fasting period before study: no
- Housing: individually in suspended stainless steel cages
- Diet (e.g. ad libitum): PMI Certified Rodent Chow® #5002 (Purina Mills, Inc.) ad libitum
- Water (e.g. ad libitum): municipal tap water ad libitum
- Acclimation period: at least seven days prior to randomisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26
- Humidity (%): 30 to 70
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: area starting at the scapulae to the hipbone and half way down the flank on each side
- Type of wrap if used: covered with a 4-ply gauze pad and the torso was wrapped with Coflex (semi-occlusive binder) that was secured with adhesive tape
- Time intervals for shavings or clipplings: 18 hours/day

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with gauze moistened with tap water
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.15 mL/animal (100 mg/kg/day group), 0.49 mL/animal (330 mg/kg/day group), 1.49 mL/ animal (control and 1000 mg/kg/day groups)
- Constant volume or concentration used: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 100, 330, 1000 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10
Control animals:
other: yes: reverse osmosis deionised (RODI) tap water
Details on study design:
- Dose selection rationale:
Dose levels for this study were selected in an attempt to produce graded responses to the test article. The high-dose level was expected to produce toxic effects, but not excessive lethality. The mid-dose level was expected to produce no or minimal observable effects. The low-dose level was expected to produce no observable effects. Dosage levels were based on data from the 14-day range-finding study conducted on this test article.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: approximately 30-90 minutes afer daily patch removal

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning and afternoon

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: approximately 30-90 minutes following daily patch removal

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the initiation of treatment on study days -2 and -1 and weekly during the study period. A final body weight was obtained from each animal prior to scheduled euthanasia (day 90).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/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: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to in-life initiation (day -2/-3) and near in-life completion (day 87 females, day 88 males)
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled euthanasia (day 90)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Following parameterswere examined: Erythrocyte count (RBC), Hematocrit (Hct), Hemoglobin concentration (Hgb), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Platelet count, Total and differential leukocyte counts, Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled euthanasia (day 90)
- Animals fasted: Yes
- How many animals: all animals
- Following parameters were examined: Alanine aminotransferase (ALT), Albumin, Albumin/globulin ratio (calcuiated), Alkaline phosphatase, Aspartate aminotransferase (AST), Calcium, Cholesterol, Blood creatinine, Gamma glutamyl transpeptidase (GGT), Globulin (calculated), Glucose, Electrolytes (sodium, potassium and chloride), Phosphorus, Total bilirubin, Total serum protein, Urea nitrogen

URINALYSIS: Yes
- Time schedule for collection of urine: day 90
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined: Bilirubin, Color and appearance, Glucose, Ketones, Leukocytes, Microscopy of spun deposits, Nitrites, Occult blood, pH, Protein, Specific gravity, Urobilinogen, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once prior to the initiation of dosing and once weekly during weeks 0-11
- Dose groups that were examined: all dose groups
- Battery of functions tested: grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Statistical analyses were performed using a MicroVax 3100 computer. The level of significance was a minimum of 5% (p <0.05) and tests were two-tailed. The summary tables indicate the level of significance detected. The control group data were compared to the data of treated groups using all groups or by a group by group comparison depending on the test. Continuous data were analysed by one-way analysis of variance (ANOVA). If significance was detected, a group by group comparison was performed using the Tukey-Kramer method. Ranked and count data were analysed by Kruskal-Wallis non-parametric ANOVA. If significance was detected, a group by group comparison was performed with Kruskal-Wallis non-parametric ANOVA. Descriptive and quantitative data were analysed by Fischer's Exact Test. If significance was detected, a group by group comparison was conducted using Fischer's Exact Test.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Minor treatment-related dermal effects did occur and included a dose-related increase in the incidence of focal pinpoint eschar, desquamation and red pinpoint areas.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
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 mortality or notable clinical abnormalities were observed during this 90-day study. The clinical findings that were noted in the treated animals were comparable to those in the controls and were considered typical of a dermal toxicity study in the rat where the animals are repeatedly wrapped.

BODY WEIGHT AND WEIGHT GAIN
Group mean body weights were generally comparable throughout this study with 1000 mg/kg/day males and females within -4% and +2%, respectively, of the controls by study termination. A few instances of statistically decreased body weight gain were noted during this study. However, these findings were not considered to be toxicologically meaningful due to their sporadic occurrence and the lack of a dose-response relationship.

FOOD CONSUMPTION
Food consumption was comparable between control and treated animals throughout this study. No statistically or toxicologically significant differences were observed.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related ocular changes occurred on this study. Several instances of slight corneal crystals and one instance of a slight anterior cataract were observed at the end of this study. However, corneal crystals are common background lesions in this strain and age of rat and were not considered toxicologically significant. Although less frequently noted, cataracts are occasionally observed in rats of this age. Therefore, since this finding only occurred in a single group 3 animal, the cataract was not considered to be treatment related.

HAEMATOLOGY
No apparent toxicologically significant abnormalities were observed in the male or female haematology and coagulation data obtained on this study. A statistically increased segmented neutrophil count was noted in the 1000 mg/kg/day females on study day 90. However, this finding was not considered meaningful due to the absence of any dose-related increases in lymphocytes or the total leukocyte count in this group.

CLINICAL CHEMISTRY
In males, statistically increased phosphorus (100, 330 and 1000 mg/kg/day groups) and calcium (1000 mg/kg/day group) were noted on study day 90. Due to the dose-related nature of these findings and the combined increases in both phosphorus and calcium (which is generally expected to occur), these changes may have been related to test article treatment. However, the biological significance of these changes is questionable since group means were within the SLI historical control range for these parameters. In addition, there were no microscopic changes in the kidneys or other correlative clinical pathology findings that would indicate that these changes were toxicologically meaningful. Potassium was also noted to be statistically increased in the 330 mg/kg/day males on day 90. However, the toxicological significance of this finding is questionable due to the lack of a dose-response relationship. In females, no statistically significant or apparent toxicologically meaningful changes were observed.

URINALYSIS
Urinalysis parameters were comparable between control and treated animals at the end of this study. No statistically or toxicologically significant differences were observed.

NEUROBEHAVIOUR
There was no apparent evidence of a neurotoxic response to the test article during this study. A few FOB parameters were noted to be statistically decreased relative to the controls at the end of this study. The most notable of these included decreased forelimb grip strength and body temperature in the 1000 mg/kg/day females. However, these changes do not appear to be a neurotoxic effect as only one parameter per functional domain was affected (out of a total of 8 to 9 parameters) with no apparent abnormalities noted in the remaining parameters. Statistically increased rearing was observed on day 5 (100 mg/kg/day females) and statistically decreased rearing was observed on day 11 (330 mg/kg/day females). In addition, statistically decreased landing foot splay (1000 mg/kg/day males) was observed at the end of this study. However, these findings were not considered to be toxicologically meaningful since there was no dose-response relationship with regard to the rearing finding and decreases in landing foot splay are not considered to be toxicologically meaningful.

ORGAN WEIGHTS
No statistically significant or toxicologically meaningful changes were observed in the absolute or relative organ weight data.

GROSS PATHOLOGY
There were no apparent treatment-related gross necropsy findings at scheduled euthanasia on study day 90. All observations are commonly observed in this species and study type and were also consistent with the controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test article-related microscopic lesions were observed following histopathological evaluation of the organs/tissues. Most microscopic findings were typical of spontaneous findings in clinically normal rats of this strain and age. In one control female, a hepatocellular adenoma was observed. Although it is not common to find a liver adenoma in this age of rat, this finding does occur sporadically in untreated animals and was not considered to be treatment related since it occurred in a control animal.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, dermal administration of hydroxyethyl urea (based on the test substance EXP 3982 (N-2-hydroxyethylurea)) at up to 1000 mg/kg/day for 90 consecutive days did not produce mortality or toxicologically meaningful effects on behaviour or weight gain in the rat and did not induce any histopathological changes in the organs and tissues. The dermal dose of 1000 mg/kg/day (as hydroxyethyl urea) is the no-observed-adverse-effect level (NOAEL) for male and female rats in this 90-day repeated dermal dose study.
Executive summary:

In the GLP and EPA Guideline OPPTS 870.3250 conform study, 10 male and 10 female Sprague-Dawley rats per group were exposed to 0 (RODI water), 100, 330 and 1000 mg/kg bw/day of hydroxyethyl urea (based on the test EXP 3982 (N-2 -hydroxyethylurea)) via the dermal route for 90 consecutive days. The test material was spread evenly over the treated skin. The test site was then covered with a 4-ply gauze pad and the torso was wrapped with an elastic wrap (semi-occlusive binder) that was secured with adhesive tape. The dorsal skin of each animal was clipped prior to dosing on day -1 and as necessary thereafter. After approximately six hours of exposure each day, the dermal wraps were removed and the test sites were washed with gauze moistened with tap water. Cage-side observations were performed for each rat approximately 30-90 minutes after patch removal. Application sites were examined daily approximately 30-90 minutes following patch removal for erythema, oedema and other changes. An abbreviated functional observation battery (home cage, removal from home cage and open field) was performed once prior to the initiation of dosing (day -1) and weekly during weeks 0-11 for each animal. A full FOB assessment (home cage, removal from home cage, open field, manipulative test and motor activity) was performed during week 12. Individual body weights were recorded twice prior to in-life initiation, weekly thereafter and prior to scheduled euthanasia. Food consumption was recorded weekly on the same day as body weights during the study. Blood and urine samples were obtained from all animals on the day of scheduled euthanasia (day 90) for evaluation of selected clinical pathology parameters. Ophthalmology examinations were performed on all animals prior to the initiation of the study and near the end of the study. Each rat was subjected to a full gross necropsy examination at the time of scheduled euthanasia (day 90). Fresh organ weights were obtained for all animals at scheduled euthanasia and selected tissues were preserved from all rats. All tissue and organs collected at necropsy from all animals in the control and high-dose groups and the liver, kidneys, spleen, testes and gross lesions from all animals in the low- and mid-dose groups were examined microscopically.

Results: Dermal administration of EXP 3982 (N-2-hydroxyethylurea) for 90 consecutive days did not produce mortality, notable clinical abnormalities or evidence of a neurotoxic response. In addition, no toxicologically meaningful changes were noted with regard to body weight gain, food consumption, urinalysis, ophthalmology, haematology, coagulation, gross necropsy or organ weight data.

Repeated dermal application of the test article did not produce significant lesions at the dermal test sites, although, minor treatment-related dermal effects were observed during the study. These included a dose-related increase in the incidence of focal/pinpoint eschar, desquamation and red pinpoint areas with a slightly higher incidence in females. However, the lack of progression of the dermal irritation and the absence of any microscopically observable changes at the test site following histopathological evaluation indicates that the above changes were superficial and had no substantive impact on the skin. Statistically increased phosphorus (100, 330 and 1000 mg/kg/day groups) and calcium (1000 mg/kg/day group) were noted on study day 90 in males. Due to the dose-related nature of these findings and the combined increases in both phosphorus and calcium (which is generally expected to occur), these changes may have been related to test article treatment. However, the biological significance of

these changes is questionable since group means were within the SLI historical control range for these parameters and there were no microscopic changes in the kidneys or other correlative clinical pathology findings. No test-article related microscopic lesions were observed following histopathological evaluation of the organs/tissues. Microscopic findings were generally typical of spontaneous findings in clinically normal rats of this strain and age.

Conclusion: Based on the results of this study, a dermal no-observe-adverse effect level (NOAEL) of 1000 mg/kg/day for hydroxyethyl urea (based on the test material EXP 3982 (N-2-hydroxyethylurea)) in male and female rats is proposed. There were no adverse systemic effects seen so no classification for STOT based on CLP/GHS guidelines in required.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
We have a Klimisch 1 validity 90 dermal application study in rats, which showed no indications of toxicity even at the limit dose of 1000mg/kg bodyweight. Using this NOEL as a NOAEL for the calculation of DNELS adds an addition degree of safety.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
We have a Klimisch 1 validity 90 dermal application study in rats, which showed no indications of toxicity even at the limit dose of 1000mg/kg bodyweight and only minimal local irritant effects in the skin.

Additional information

The only repeat dose study available is a 90 day dermal study using a 57.58% solution of hydroxyethyl urea in water (EXP3982). Skin contact is the most likely route of exposure as the hydroxyethyl urea is manufactured as a solution in water so this is an appropriate study. The study is Klimisch 1 validity. REACH guidance allows calculation of oral and inhalation DNELs from dermal data on a cases by case basis. Model data (Epiwin) indicates oral absorption in the region of 77%, dermal absorption is also predicted to be 78.3% into the viable epidermis and therefore available for absorption into the systemic circulation. When using oral data it is accepted to calculate dermal DNELs, but in general the dermal absorption would be expected to be lower, however as no adverse effects were seen at the 1000mg/kg bodyweight in the dermal study this represent a NOEL, with the true NOAEL likely to be significantly higher and the NOAEL also higher still. The Epiwin model data indicates a high level of both oral and dermal absorption both in the region of over 70% which justifies the use of the dermal NOAEL to calculate ORAL and inhalation long term DNELs. Therefore using the dermal 1000mg/kg bodyweight as an oral NOAEL is unlikely to underestimate the toxic potential. As for the inhalation route, again we are starting from a very conservative NOAEL converted to a NOAEC. Also as recommended by ECHA an additional factor of two will be applied, so assuming 100% absorption in the lungs and 50% via the skin. It is considered that there are sufficiently conservative assumptions built into these calculations to ensure safe DNELs can be calculated.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
There is no repeat dose oral toxicity study for hydroxyethyl urea, there is Klimisch 1 validity 90 day dermal toxicity study on a 57.58% solution of hydroxyethyl urea (EXP3982) which can be used to define DNELs. Available physical and chemical information suggest that hydroxyl ethyl urea will be absorbed through the skin so this study can be used for the derivation of DNELs. The Epiwin model data indicate ca. 77% oral absorption and ca.78% dermal absorption which supports the use of the dermal NOAEL when calculating the oral DNELs. The date from the Epiwin model and the lack of any evidence of toxic effects from the acute oral and dermal toxicity studies supports the use of the dermal data.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
We do not have a repeated dose in halation study on hydroxyethyl urea. We do have a high quality dermal application study on a 57.58% solution of hydroxyethyl urea (EXP3982). Inhalation is not expected to be a significant route of exposure as the hydroxyethyl urea is manufactured as a solution in water and is not dried to a powder. It has a low vapour pressure so exposure to vapour from the aqueous solution is not expected. It is also not expected that any fine aerosols would be generated in it's handling and use. We have an acute inhalation study on a product containing hydroxyethyl urea showing no indication of toxic effect at 4mg/l the highest level tested. When extrapolating from oral to inhalation exposure an additional factor of 2 is recommended by ECHA, to calculate an inhalation long term systemic DNEL. This assumes 100% absorption by inhalation and 50% by the oral route. The Epiwin model predicts ca. 77% oral and 78% dermal absorption, therefore using the ECHA 50% assumption is acceptable. It is considered that the additional factor of two as used when using an oral NOAEL should be sufficient in setting an inhalation long term DNEL. In addition the starting point is the dermal 1000mg/kg limit dose at which no adverse effects were seen, rather than for a true NOAEL. This will result in an adequately conservative DNEL. Based on this, using the REACH guidance a DNEL for systemic effects from inhalation can be calculated for the dermal highest dose which was a NOEL. Therefore it is not scientifically justified to carry out an inhalation study; this avoids the use of additional animals.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
We have an acute inhalation toxicity study with exposure up to 4mg/l of hydroxyethyl urea; this did not show any specific indications of local effect such as irritation. Some red spots were seen on the lungs of some animals, but when examined histopathologically these were not confirmed as being related to treatment.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The dermal route is the most likely route of exposure during the manufacture and use of hydroxyethyl urea, which is always manufactured and handled as a solution in water. We have a Klimisch 1 validity 90 dermal application study in rats, which showed no indications of toxicity even at the limit dose of 1000mg/kg bodyweight. The Epiwin model data indicates that hydroxyethyl urea would be absorbed through the skin at ca 78%. This study is therefore suitable for use to calculate a long-term dermal DNEL, based on the NOEL of 1000mg/kg bodyweight which can be considered to be the NOAEL which will add an addition margin of safety to the DNEL.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
We have information from the 90 day dermal study which only showed minimal dermal irritation but showed no histopathological changes in the skin at the 1000mg/kg bodyweight, limit dose. It is therefore not necessary to perform any additional animal test for local dermal effect of exposure.

Justification for classification or non-classification

Classification for Specific Target Organ Toxicity STOT, by the EU CLP (GHS) guideline requires serious adverse effects on organs at dose levels of less than 100mg/kg bodyweight/day in a 90 day study. As no adverse effects were seen in the 90 dermal study at the 1000mg/kg limit dose 10 times higher than the upper limit for STOT classification there is no requirement for a classification for STOT effects. Therefore there were no effects that would require classification for STOT according to the EU CLP guidelines.