Oligomerisation products of sucrose with ethylene oxide and methyloxirane

Endpoint summary

Description of key information

NOAEL (28 days repeated dose, Wistar rats): ≥ 1000 mg/kg bw.

NOEL (90 days repeated dose, F344/DuCrl rats): 300 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Follows GLP and OECD guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

GLP characterization of the test material was done concurrently with the study.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

yes

Remarks:

GLP characterization of the test material was done concurrently with the study.

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

GLP characterization of the test material was done concurrently with the study.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: F344/DuCrl

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Kingston, New York)
- Age at study initiation: Approximately 7 weeks
- Housing: After assignment, animals were housed two per cage in stainless steel cages. Cages had solid floors with corncob bedding and paper nesting material for enrichment. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Animals were acclimated to the laboratory for at least one week prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
All dosing solutions were prepared by mixing the test material in ultrapure water at concentrations of 25, 75, or 250 mg/ml and administered at a dose volume of 4 ml/kg body weight to achieve the targeted dose levels. Dose solutions were not corrected for purity. Dose volumes were adjusted at least weekly based on individual body weights. The control rats were dosed with ultrapure water at 4 ml/kg body weight. Dose solutions were prepared periodically throughout the study.

VEHICLE
De-ionized water used to prepare the test solutions was purified through a PURELAB Ultra water treatment system (ELGA LabWater, High Wycombe, United Kingdom) producing ultrapure water.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Confirmation and Homogeneity
Dose confirmation analyses of all dose solutions were determined pre-exposure, near the middle, and near the end of the study. The homogeneity of the low-dose and the high-dose test solutions was determined concurrent with dose confirmation. The method used for analyzing the test material in ultrapure water was liquid chromatography-mass spectrometry (LC/MS).

Stability
A previously conducted stability study (McFadden and Hales, 2015) showed Ethylenediamine, ethoxylated and propoxylated (>1 – <8.5 mol of EO and PO) to be stable for at least 24 days in ultrapure water, at dose levels ranging from 0.25 to 250 mg/ml. The established concentration range and duration spanned those used in this study; therefore, additional stability analyses were not conducted.

Retainer Samples
Retained samples (one/dose/mix plus control) were stored in sealed vials in a manner consistent with the sample retention policy of the laboratory.

Duration of treatment / exposure:

90-days

Frequency of treatment:

once daily seven days/week

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A possible route of human exposure to the test material would be via accidental ingestion. Thus, oral administration of the test material to rats via oral gavage represented an appropriate means of exposure. Animals were administered Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) once daily seven days/week for at least 90 days.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Detailed clinical observations (DCO) were conducted on all animals pre-exposure and once per week throughout the study

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed pre-exposure and weekly throughout the dosing period. Body weight gains were calculated relative to day 1.

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined by a veterinarian pre-exposure and prior to the
scheduled necropsy using indirect ophthalmoscopy.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained from the orbital sinus following anesthesia via inhalation of O2/isoflurane at the scheduled necropsy.
- Anaesthetic used for blood collection: Yes (O2/isoflurane)
- Animals fasted: Yes-- overnight prior to blook collection
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected and serum was separated from cells as soon as possible. Serum parameters were measured using a cobas c311 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Indiana).
- Animals fasted: Yes
- How many animals: All

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine: Urine samples were obtained from all animals the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water were available during this procedure.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, organ weights, clinical chemistry data, urinalysis data, and appropriate hematologic data were evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA). If significant at alpha = 0.05, the ANOVA was followed respectively by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control. The experiment-wise alpha level was reported for these two tests. DCO incidence data (scored observations only) were statistically analyzed by a z-test of proportions comparing each treated group to the control group (alpha = 0.05; Bruning and Kintz, 1987). Data collected at different time points were analyzed separately. Descriptive statistics only (means and standard deviations) were reported for body weight gains, RBC indices, and differential WBC counts. Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969), but routinely excluded only from feed consumption calculations. Outliers may have been excluded from other analyses only for documented, scientifically sound reasons.
If deemed necessary, more statistical tests were performed. Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses, along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.

Clinical signs:

no effects observed

Description (incidence and severity):

All rodents survived the 90-day test period. There were no treatment-related clinical findings or scored DCO observations in males or females given Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) at any dose level.

Mortality:

no mortality observed

Description (incidence):

All rodents survived the 90-day test period. There were no treatment-related clinical findings or scored DCO observations in males or females given Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) at any dose level.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no differences in the body weights of male or female rats when compared to their respective controls and no differences related to treatment of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatmentrelated differences in feed consumption between any of the treatment groups as compared to their respective controls.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmologic examinations indicated all rats were within normal limits at pre-exposure and prior to study termination.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females administered 1000 mg/kg/day of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) had statistically significant lower red blood cell counts, hemoglobin concentrations and hematocrits.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment-related alterations in any of the clinical chemistry parameters for male and female rats at any dose level.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related alterations in the urinalysis parameters of male and female rats at any dose level.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related alterations in organ weights of male and female rats at any dose level.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross pathologic observations in males or females at any dose level.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY: There were no treatment-related clinical findings or scored DCO observations in males or females given Ethylenediamine,
ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) at any dose level. All rodents survived the 90-day test period.

BODY WEIGHT AND WEIGHT GAIN: There were no differences in the body weights of male or female rats when compared to their respective controls and no differences related to treatment of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): There were no treatmentrelated differences in feed consumption between any of the treatment groups as compared to their respective controls. There were a few sporadic occurrences of statistically significant, slightly higher or lower feed consumption in males and/or females in all of the treatment groups as compared to their respective controls. These differences were interpreted to be unrelated to treatment with Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) due to their spurious nature and/or lack of a dose-response relationship.

OPHTHALMOSCOPIC EXAMINATION: Ophthalmologic examinations indicated all rats were within normal limits at pre-exposure and prior to study termination.

HAEMATOLOGY: Males and females administered 1000 mg/kg/day of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) had statistically significant lower red blood cell counts, hemoglobin concentrations and hematocrits, which were interpreted to be treatment-related. These lower erythrocytic parameters were accompanied by the histopathologic observation of very slight increased erythrocytic extramedullary hematopoiesis of the spleen in 8/10 males administered 1000 mg/kg/day of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO). The increased splenic extramedullary hematopoiesis in males administered 1000 mg/kg/day was interpreted to be a compensatory response to the minor decrements in erythrocytic parameters. Females did not have a corresponding treatment-related increase in erythrocytic extramedullary hematopoiesis of the spleen at any dose level. Females administered 100 or 300 mg/kg/day of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) had statistically significant lower red blood cell counts, which were interpreted to be unrelated to treatment due to the lack of a dose-responsive progression of the decrements at these dose levels, and the absence of a histological correlate and treatment-related changes on other hematologic parameters.

CLINICAL CHEMISTRY: There were no treatment-related alterations in any of the clinical chemistry parameters for male and female rats at any dose level. Males administered 1000 mg/kg/day of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) had a statistically significant higher phosphorus concentration. Males and females administered 1000 mg/kg/day had statistically significant lower sodium concentrations, and females administered 100 or 1000 mg/kg/day had statistically significant higher glucose concentrations. These alterations were interpreted to be unrelated to treatment because all of the values were within the historical control range of recently conducted studies from this laboratory. Females administered 100 mg/kg/day of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) had a statistically significant lower aspartate aminotransferase activity that was interpreted to be unrelated to treatment due to the lack of a dose response.

URINALYSIS: There were no treatment-related alterations in the urinalysis parameters of male and female rats at any dose level. Males administered 300 mg/kg/day of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) had statistically significant lower urine volume and higher specific gravity. These alterations were interpreted to be unrelated to treatment because of the lack of a dose response, and the values were within the historical control range of recently conducted studies from this laboratory.

ORGAN WEIGHTS: There were no treatment-related alterations in organ weights of male and female rats at any dose level. Males administered 300 or 1000 mg/kg/day of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) had statistically significant higher absolute kidney weights. The higher kidney weights were interpreted to be unrelated to treatment because of the lack of any effects on kidneyassociated clinical chemistry parameters, the absence of a histopathology correlate in the kidneys, and the weights were within the historical control range of recently conducted studies from this laboratory.

GROSS PATHOLOGY: There were no treatment-related gross pathologic observations in males or females at any dose level. All gross pathologic observations
were considered to be spontaneous alterations, unassociated with oral gavage administration of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO).

HISTOPATHOLOGY: NON-NEOPLASTIC
Males administered 1000 mg/kg/day of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) had a treatment-related higher incidence of increased erythrocytic extramedullary hematopoiesis as compared to controls. The increased splenic extramedullary hematopoiesis in males administered 1000 mg/kg/day was interpreted to be a compensatory response to the minor decrements in erythrocytic parameters at this dose level. The low incidence of increased extramedullary hematopoiesis (one or two rats) in females from the control group and all dose levels was considered to be unrelated to treatment.
Other treatment-related histopathologic effects were interpreted to be point-of-contact irritative lesions, located in the nasal tissues, lungs, and stomach. Numerous irritative lesions were present in the olfactory and/or respiratory epithelium of the nasal passages in some males and females from all treated dose levels. The nasal alterations were most prominent in the ethmoid turbinates, ventral wall, and/or ventral septum of the posterior aspect of the nasal passages. The most common nasal epithelial alteration was atrophy of the olfactory epithelium, characterized by focal or multifocal areas with thinning of the apical cytoplasm, loss of cilia, and disarray of the epithelial cells at the ventral and posterior tips of the ethmoid turbinates. Other irritative nasal effects, noted in lesser numbers of rats, consisted of atrophy of the respiratory epithelium, suppurative exudate, acute inflammation of the olfactory and/or respiratory epithelium, necrosis of the olfactory epithelium, and ulcers of the olfactory and/or respiratory epithelium. All of these nasal epithelial effects were consistent with entry of the test material into the posterior aspect of the nasal passages due to inadvertent reflux of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) from the esophagus or oropharynx immediately following the oral gavage dosing procedure, and were not related to systemic toxicity of the test material. One male administered 1000 mg/kg/day had slight, multifocal bronchiolo-alveolar hyperplasia with inflammation, and slight, multifocal hyperplasia of peribronchial lymphoid tissue of the lungs. The lung lesions in this rat were interpreted to be caused by inadvertent reflux of the test material into the lungs immediately following the oral gavage dosing procedure, and were not related to systemic toxicity of the test material. The stomach of males and females administered 1000 mg/kg/day also had irritative lesions, which were not related to systemic toxicity, but were due to direct contact with the test material following oral gavage administration. The most common stomach lesion was very slight or slight, focally extensive hyperplasia with inflammation of the epithelium lining the limiting ridge. The hyperplasia of the epithelial cells lining the limiting ridge was characterized by an increased number of basal epithelial cells and an increase in the number of mitotic figures in the basal region of the epithelium. Epithelial cells at all levels of the limiting ridge had increased cytoplasmic basophilia. The associated inflammation consisted of numerous neutrophils, and lesser numbers of eosinophils and lymphocytes, in the lamina propria and migrating to the intercellular spaces of the squamous epithelial cell layer. Intercellular edema accompanied the inflammation of the epithelial cell layer of the limiting ridge. Most of the males and females administered 1000 mg/kg/day had very slight, multifocal vacuolar degeneration of epithelial cells in the limiting ridge of the stomach. The vacuolar degeneration was characterized by single or multiple, clear cytoplasmic vacuoles in individual cells from all levels of the squamous epithelium lining the limiting ridge. Most of the males and females administered 1000 mg/kg/day also had very slight or slight, multifocal subacute to chronic inflammation of the glandular submucosa of the stomach. The inflammation of the glandular submucosa consisted of accumulations of neutrophils, eosinophils and lymphocytes, which also extended into the adjacent lamina propria of the stomach. All other histopathologic observations were considered to be spontaneous alterations, unassociated with oral gavage administration of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO).

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Critical effects observed:

not specified

Hematology Differences:

Sex	Males
Dose (mg/kg/day)	Historical Control^@	0	100	300	1000
Red Blood Cell Count (E6/uL)	9.16 -10.27	9.69	9.57	9.54	9.32*
Hemoglobin (g/dL)	14.9 -17.0	15.6	15.5	15.5	15.2*
Hematocrit (%)	45.6 -51.9	47.0	45.4	46.3	45.4*
Sex	Female
Dose (mg/kg/day)	Historical Control^@	0	100	300	1000
Red Blood Cell Count(E6/uL)	7.57 -9.41	8.97	8.75*	8.79*	8.61*
Hemoglobin (g/dL)	13.5 -16.8	15.4	15.2	15.2	14.9*
Hematocrit (%)	39.4 -49.4	45.9	45.3	45.3	44.4*

@Historical control data obtained from sixteen 90-day data sets conducted in this laboratory from 2010 through 2015.

* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Bold type indicates the effects were interpreted to be treatment-related.

Clinical Chemistry Differences

Sex	Males
Dose (mg/kg/day)	Historical Control^@	0	100	300	1000
Phosphorus (mg/dL)	7.2 -10.3	7.1	7.3	7.3	7.8*
Sodium (mmol/L)	143 -152	145	145	144	144*
Sex	Females
Dose (mg/kg/day)	Historical Control^@	0	100	300	1000
Aspartate Aminotransferase(U/L)	79 -103	91	77^$	92	82
Glucose (mg/dL)	73 -137	96	155*	106	117*
Sodium (mmol/L)	143 -151	146	145	145	144*

@Historical control data obtained from sixteen 90-day data sets conducted in this laboratory from 2010

through 2015.

* Statistically different from control mean by Dunnett’s test, alpha = 0.05.

$ Statistically different from control mean byWilcoxon’s test, alpha = 0.05.

Conclusions:

Based on the treatment-related point-of-contact irritative lesions in the nasal tissues that were present in some males and females from all treated dose levels, the overall noobserved- effect level (NOEL) for F344/DuCrl rats of either sex was not determined. However, the only treatment-related effects indicative of systemic toxicity were lower red blood cell counts, hemoglobin concentrations and hematocrits in males and females administered 1000 mg/kg/day, with compensatory increased erythrocytic extramedullary hematopoiesis of the spleen in males administered 1000 mg/kg day. Therefore, the NOEL for systemic toxicity was 300 mg/kg/day Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO).

Executive summary:

The purpose of this study was to evaluate the potential toxicity of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) in rats following oral administration for at least 90 days.

Ten male and ten female F344/DuCrl rats per group were administered 0, 100, 300, or 1000 milligrams Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) per kilogram body weight per day (mg/kg/day) in ultrapure water via oral gavage for at least 90 days to evaluate the potential for systemic toxicity. Parameters evaluated were daily cage-side observations, daily clinical observations, weekly detailed clinical observations, ophthalmic examinations, body weights, feed consumption, hematology, coagulation parameters, urinalysis, clinical chemistry, selected organ weights, and gross and

histopathologic examinations.

There were no treatment-related effects in cage-side or clinical observations, weekly detailed clinical observations, body weights, feed consumption, ophthalmic examinations, clinical chemistry, prothrombin time or urinalysis parameters. There were no treatment related organ weight effects, and no treatment-related gross pathologic observations.

Males and females administered 1000 mg/kg/day had statistically significant lower red blood cell counts, hemoglobin concentrations and hematocrits, which were interpreted to be treatment-related. These lower erythrocytic parameters were accompanied by the

histopathologic observation of very slight increased erythrocytic extramedullary hematopoiesis of the spleen in 8/10 males administered 1000 mg/kg/day. The increased splenic extramedullary hematopoiesis in males administered 1000 mg/kg/day was

interpreted to be a treatment-related compensatory response to the minor decrements in erythrocytic parameters. Females did not have a corresponding increase in erythrocytic extramedullary hematopoiesis of the spleen at any dose level.

Other treatment-related histopathologic effects were interpreted to be point-of-contact irritative lesions, located in the nasal tissues, lungs and stomach. Numerous irritative lesions were present in the olfactory and/or respiratory epithelium of the nasal passages in some males and females from all treated dose levels. The nasal alterations were most prominent in the ethmoid turbinates, ventral wall, and/or ventral septum of the posterior aspect of the nasal passages. The most common nasal epithelial alteration was atrophy of the olfactory epithelium. Other irritative nasal effects, noted in lesser numbers of rats, consisted of atrophy of the respiratory epithelium, suppurative exudate, acute inflammation of the olfactory and/or respiratory epithelium, necrosis of the olfactory epithelium, and ulcers of the olfactory and/or respiratory epithelium. All of these nasal epithelial effects were consistent with entry of the test material into the posterior aspect of the nasal passages due to inadvertent reflux of the test material from the esophagus or oropharynx immediately following the oral gavage dosing procedure, and were not related to systemic toxicity of the test material. One male administered 1000 mg/kg/day had slight, multifocal bronchioloalveolar hyperplasia with inflammation, and slight, multifocal hyperplasia of peribronchial lymphoid tissue of the lungs. The lung lesions in this rat were interpreted to be caused by inadvertent reflux of the test material into the lungs immediately following the oral gavage dosing procedure, and were not related to systemic toxicity of the test material. The stomach of males and females administered 1000 mg/kg/day also had irritative lesions which were not related to systemic toxicity, but were due to direct contact with the test material following oral gavage administration. The most common stomach lesion was very slight or slight, focally extensive hyperplasia with inflammation of the epithelium lining the limiting ridge.

Most of the males and females administered 1000 mg/kg/day also had very slight, multifocal vacuolar degeneration of epithelial cells in the limiting ridge of the stomach, and very slight or slight, multifocal subacute to chronic inflammation of the glandular submucosa of the stomach.

Based on the treatment-related point-of-contact irritative lesions in the nasal tissues that were present in some males and females from all treated dose levels, the overall noobserved- effect level (NOEL) for F344/DuCrl rats of either sex was not determined. However, the only treatment-related effects indicative of systemic toxicity were lower red blood cell counts, hemoglobin concentrations and hematocrits in males and females administered 1000 mg/kg/day, with compensatory increased erythrocytic extramedullary hematopoiesis of the spleen in males administered 1000 mg/kg day. Therefore, the NOEL for systemic toxicity was 300 mg/kg/day Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content of the test item was assumed to be nominally 100% for calculation.
Before the start of the study formulations containing the test item in concentrations
of 2 mg/mI and 200 mg/ml were analyzed to determine homogeneous distribution
(not for the 2mg/ml formulation as it was a solution), content and stability of the test
item in tap water. During the study homogeneity and content was checked twice.

Duration of treatment / exposure:

31 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

five

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels used were based on the results of a pilot study. During this study Wistar rats (2 males and 2 females per dose group) received the test substance in doses of O, 65, 160, 400, 1000 mg/kg for a period of two weeks. No clinical findings or relevant effects on body weight development were observed in this study.

Positive control:

no positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes (incl. Open Field Observations (OFO))
- Time schedule: day 8, 15, 22, 28

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: YES
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once, day 30
- How many animals: all dose groups incl. controls

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once, day 30
- How many animals: all dose groups incl. controls

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: FOB: once, day 25; MA: once, day 29
- Dose groups that were examined: all dose groups incl. controls
- Battery of functions tested: Functional Observational Battery (FOB); Motor Activity (MA)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (all dose groups and controls)

ORGAN Weights:
Adrenals, brain, epididymides, heart, kidneys, Liver, Ovaries, Spleen, Testes, Thymus, uterus

Fixed organs:
Adrenals, aorta, brain (cerebrum, cerebellum, ponslmedulla), epididymides, esophagus, eyes, eyelids, extraorbital lachrymal glands, femur, harderian glands, head (with nasal and paranasal cavities), heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum and remaining intestine), kidneys, larynx, liver, lymph nodes (mandibular, bronchial/hilus, and mesenteric), lung, mamma, optical nerves, ovaries, oviducts, pancreas, pharynx, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulating glands), skeletal muscle (thigh), skin (mammary and muzzle), spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach (forestomach and glandular stomach), testes, thymus, thyroids (including parathyroid glands), tongue, trachea, ureter, urethra, urinary bladder, uterus with uterine cervix, vagina, Zymbal’s glands and all organs or tissues with macroscopic findings.

HISTOPATHOLOGY: Yes (high dose group and controls)
- Microscopic: Adrenals, brain (cerebrum, cerebellum, ponslmedulla), epididymides, femur, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum and remaining intestine), kidneys, liver, lung, lymph nodes, ovaries, oviducts, pancreas, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulating glands), skeletal muscle (thigh), skin (mammary and muzzle), spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach (forestomach and glandular stomach), testes, thymus, thyroids (including parathyroid glands), trachea, urinary bladder, uterus with uterine cervix, and all organs or tissues with macroscopic findings. Slides were prepared from the first five animals of all groups and evaluated from the control and the high concentration group.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Haematology:

Differential blood count revealed as the only conspicuous finding, that the number of leucocytes and lymphocytes was highest in high dose males but was lowest in high dose females. However, these differences were slight compared to the differences between the means of historical control values and the upper 2 s-range in males and the lower 2s-range in females, respectively. Furthernore, the deviations from control value were in opposite directions in males and females and histopathological examinations revealed no corresponding findings. For these
reasons a toxicological relevance was not inferred from these data.

Clinical chemistry:
Clinical aboratory tests revealed a significantly increased activity of alanine aminotransferase in females at 1000 mg/kg. However, the difference to control was relatively slight and histopathological examination of the liver produced no evidence for a treatment-related effect. Therefore, a toxicological relevance is not assumed. Determination of substrates showed the plasma creatinine concentration in males at 1000 mg/kg to be significantly decreased and in all treated female groups to be significantly increased. However, the differences to the respective control value were slight, no dose dependence was present in females, the deviation from the respective control group pointed in opposite directions in males and females and histopathological examination of kidneys produced no corresponding finding. On the basis of these results this was was not considered to be of toxicological relevance.

Histopathology:
The histopathological evaluation revealed a slightly more pronounced hypertrophy of the follicular cell epithelium in the thyroid gland of fernales ofthe high dose, in which not only centrally located follicles appeared hypertrophic but also follicles of the periphery. Hypertrophy was observed to a minimal degree also in females of the other dose groups including one control animal. Due to the slightly increased severity grade in females ofthe high dose group, an effect of the test substance cannot be completely ruled out. Such minimal changes are regarded to be caused by substances that interfere with general metabolic processes and are so far assessed as indirect and adaptive effects. If at all the findings in the present study were related to the treatment, they are therefore not considered as a primary adverse effect on thyroids. However, due to the missing dose response (incidence for follicular cell hypertrophy was: 1 /3/ 1 /3) and the lack of any other treatment-related changes a spontaneous occurrence e.g. by a general variability seems more likely.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

not specified

Conclusions:

The NOAEL for the given test substance was 1000 mg/kg bw/day.

Executive summary:

In a repeated dose oral toxicity study in rats (Wistar, OECD TG 407), 2,2',2" -Nitrilotriethanol, propoxylated was adiministered via gavage to 5 rats/sex/dose at 0, 100, 300, 1000 mg/kg bw for 4 weeks.. No death was observed in either sex. No clinical effects were observed in both sex of all dose groups.There was no effect observed upon haematological, clinical biochemistry or macroscopic examination at any dose. Based on these results the NOAEL was considered to be 1000 mg/kg bw/day. If at all the slight changes in thyroids of females at 1000 mg/kg were related to the treatment, they are regarded as an indirect and adaptive effect.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Results 'read across' from study on 2, 2', 2''-nitrilotriethanol, propoxylated, as permitted by Annex XI paragraph 1.5, based on the justification in the report by Paul Illing Consultancy Services Ltd and Marlin Consultancy (Illing and Barratt, 2007). The report identifies that 2, 2', 2''-nitrilotriethanol, propoxylated is the most bioavailable of the NLP polyols linked by an ether group, and the lack of toxicity seen for it and for both components (sucrose and -propane-1,2-diol) of sucrose, ethoxylated and propoxylated should be considered representative of of the lack of toxicity of the ether linked polyol. Thus, on animal welfare grounds this test should not be undertaken on sucrose, ethoxylated and propoxylated. For further details concerning the grouping, consult Illing and Barratt, 2007.

In a repeated dose oral toxicity study in rats (Wistar, OECD TG 407), 2,2',2" -Nitrilotriethanol, propoxylated was adimistered via gavage to 5 rats/sex/dose at 0, 100, 300, 1000 mg/kg bw for 4 weeks.. No death was observed in either sex. No clinical effects were observed in both sex of all dose groups.There was no effect observed upon haematological, clinical biochemistry or macroscopic examination at any dose. Based on these results the NOAEL was considered to be ≥ 1000 mg/kg bw/day. If at all the slight changes in thyroids of females at 1000 mg/kg were related to the treatment, they are regarded as an indirect and adaptive effect.

 

A subchronic oral toxicity study is available for the 'read-across' substance EDA EO PO:

The purpose of this study was to evaluate the potential toxicity of Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) in rats following oral administration for at least 90 days.

Ten male and ten female F344/DuCrl rats per group were administered 0, 100, 300, or 1000 milligrams Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) per kilogram body weight per day (mg/kg/day) in ultrapure water via oral gavage for at least 90 days to evaluate the potential for systemic toxicity. Parameters evaluated were daily cage-side observations, daily clinical observations, weekly detailed clinical observations, ophthalmic examinations, body weights, feed consumption, hematology, coagulation parameters, urinalysis, clinical chemistry, selected organ weights, and gross and

histopathologic examinations.

There were no treatment-related effects in cage-side or clinical observations, weekly detailed clinical observations, body weights, feed consumption, ophthalmic examinations, clinical chemistry, prothrombin time or urinalysis parameters. There were no treatment related organ weight effects, and no treatment-related gross pathologic observations.

Males and females administered 1000 mg/kg/day had statistically significant lower red blood cell counts, hemoglobin concentrations and hematocrits, which were interpreted to be treatment-related. These lower erythrocytic parameters were accompanied by the

histopathologic observation of very slight increased erythrocytic extramedullary hematopoiesis of the spleen in 8/10 males administered 1000 mg/kg/day. The increased splenic extramedullary hematopoiesis in males administered 1000 mg/kg/day was

interpreted to be a treatment-related compensatory response to the minor decrements in erythrocytic parameters. Females did not have a corresponding increase in erythrocytic extramedullary hematopoiesis of the spleen at any dose level.

Other treatment-related histopathologic effects were interpreted to be point-of-contact irritative lesions, located in the nasal tissues, lungs and stomach. Numerous irritative lesions were present in the olfactory and/or respiratory epithelium of the nasal passages in some males and females from all treated dose levels. The nasal alterations were most prominent in the ethmoid turbinates, ventral wall, and/or ventral septum of the posterior aspect of the nasal passages. The most common nasal epithelial alteration was atrophy of the olfactory epithelium. Other irritative nasal effects, noted in lesser numbers of rats, consisted of atrophy of the respiratory epithelium, suppurative exudate, acute inflammation of the olfactory and/or respiratory epithelium, necrosis of the olfactory epithelium, and ulcers of the olfactory and/or respiratory epithelium. All of these nasal epithelial effects were consistent with entry of the test material into the posterior aspect of the nasal passages due to inadvertent reflux of the test material from the esophagus or oropharynx immediately following the oral gavage dosing procedure, and were not related to systemic toxicity of the test material. One male administered 1000 mg/kg/day had slight, multifocal bronchioloalveolar hyperplasia with inflammation, and slight, multifocal hyperplasia of peribronchial lymphoid tissue of the lungs. The lung lesions in this rat were interpreted to be caused by inadvertent reflux of the test material into the lungs immediately following the oral gavage dosing procedure, and were not related to systemic toxicity of the test material. The stomach of males and females administered 1000 mg/kg/day also had irritative lesions which were not related to systemic toxicity, but were due to direct contact with the test material following oral gavage administration. The most common stomach lesion was very slight or slight, focally extensive hyperplasia with inflammation of the epithelium lining the limiting ridge.

Most of the males and females administered 1000 mg/kg/day also had very slight, multifocal vacuolar degeneration of epithelial cells in the limiting ridge of the stomach, and very slight or slight, multifocal subacute to chronic inflammation of the glandular submucosa of the stomach.

Based on the treatment-related point-of-contact irritative lesions in the nasal tissues that were present in some males and females from all treated dose levels, the overall noobserved- effect level (NOEL) for F344/DuCrl rats of either sex was not determined. However, the only treatment-related effects indicative of systemic toxicity were lower red blood cell counts, hemoglobin concentrations and hematocrits in males and females administered 1000 mg/kg/day, with compensatory increased erythrocytic extramedullary hematopoiesis of the spleen in males administered 1000 mg/kg day. Therefore, the NOEL for systemic toxicity was 300 mg/kg/day Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO).

Justification for selection of repeated dose toxicity via oral route

- systemic effects endpoint: valid studies, suitable for assessment.

Justification for classification or non-classification

Based on the results of a valid OECD TG 407 study conducted with the 'read-across' substance 2,2',2''-Nitrilotriethanol, propoxylated with male and female Wistar rats, a NOAEL of 1000 mg/kg bw/day, for males and females, respectively, could be assessed for the oral route. Furthermore, a valid OECD TG 408 study with the 'read-across' substance EDA EO PO showed a NOEL of 300 mg/kg bw/day in rats.

Thus, there is no indication for classification/labelling of the the test item according to either the EU Directive 67/548/EEC or the CLP Regulation (EC) No. 1272/2008.