Oligomerisation products of 2,2'- oxydiethanol with ethylene oxide and propylene oxide

Endpoint summary

Description of key information

For assessment of repeated dose toxicity, the test item 2,2'-Oxybisethanol, ethoxylated and propoxylated (>1 < 4.5 mol EO and >1 <4.5 mol PO) was administered to male and female Wistar rats via the diet at dose levels of 0, 1200, 4000 and 12000 ppm (977.5, 330.5, 96 mg/kg bw) for 28 days. The test was conducted according to the OECD 407 (2008) under GLP conditions (BASFSE30C0042/11C117, 2012). The NOAEL, was set at 12000 ppm for both male and female Wistar rats, corresponding respectively to 977.5 and 1014.6 mg/kg bw/day.

Additionally, a 90 -day study with the 'read-across' substance Ethylenediamine, ethoxylated and propoxylated (>1 - <8.5 mol of EO and PO) (EDA EO PO) in F344/DuCrl rats was performed according to OECD TG 408. Rats were treated with 100, 300 and 1000 mg/kg bw/day for 90 days. A NOEL of 300 mg/kg bw/day regarding systemic toxicity was set.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Jan 2012 - 20 Jul 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 03 Oct 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

(Commission Regulation (EC) No 440/2008)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain as described in the report: Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Age at study initiation: 42 +/- 1 days
- Mean body weight at test initiation: males, 160 - 175 g; females, 120 - 140 g
- Housing: five/cage, in polysulfate cages (TECNIPLAST, Hohenpeißenberg, Germany; floor area 2065 cm²), with dust-free wooden bedding and wooden
gnawing blocks (Typ NGM E-022; Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) as environmental enrichment
- Diet: ground Kliba mouse/rat maintenance diet “GLP”, meal (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

ANALYSIS OF FOOD, WATER, BEDDING
- The food used in the study was assayed for chemical and microbial contaminants according to the Fed. Reg. Vol. 44, No. 91 of May. 09, 1979, p 27354 (EPA). Food was found to be suitable.
- The drinking water was assayed for chemical contaminants according to the German Drinking Water Regulation. Drinking water was found to be suitable.
- The bedding and enrichments were assayed for contaminants (chlorinated hydrocarbons and heavy metals). The values given in Lab Animal, Nov.–Dec. 1979, pp 24–33, serve as the guideline for maximum tolerable contaminants. Bedding and enrichments were found to be suitable.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

the test material was added to the diet

Details on oral exposure:

DIET PREPARATION
For each dose level, the test substance was weighted out and mixed with a small amount of diet. Then corresponding amounts of diet, depending on test group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The analyses of the test preparations were carried out at the Competence Center Analytics of BASF SE, Ludwigshafen/Rhein, Germany (GLP compliant).
The stability of the test substance in the diet was checked before the start of the treatment. Stability analysis proved that the test item was stable in the used carrier for 10 days at room temperature (BASF SE, Report No. 11L00456, included in the study report).
- Homogeneity of the test substance distribution within the carrier (i.e., the diet), was checked before the start of the treatment, from samples taken from the lowest and highest test concentration. Homogeneity control analysis proved that the test item was homogenously distributed in the carrier; the standard deviation for homogeneity was in the range of 3.7 to 4.9 mg/kg ((BASF SE, Report No. 12L00026, included in the study report).
- Concentration control analyses of the test preparations were performed in samples of all concentrations at the start of the administration period. The analyses revealed that the values were in the expected range of the target concentrations, i.e. 90% - 110% of the nominal concentration in all test preparations. Thus, the mean concentration of the test substance reached 100.7% - 103.9% of the nominal concentrations (BASF SE, Report No. 12L00026, included in the study report).

Duration of treatment / exposure:

28 day exposure

Frequency of treatment:

daily, seven days/week

Remarks:

Doses / Concentrations:
1200, 4000, 12000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
96, 330.5, 977.5 mg/kg bw/day for males
Basis:
actual ingested

Remarks:

Doses / Concentrations:
104.5, 332.5, 1014.6 mg/kg bw/day for females
Basis:
actual ingested

No. of animals per sex per dose:

Control (diet without test item): 5 animals/sex
Low dose (1200 ppm): 5 animals/sex
Mid dose (4000 ppm): 5 animals/sex
High dose (12000 ppm): 5 animals/sex

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

MORTALITY AND CLINICAL OBSERVATIONS
Check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. Moribund animals were sacrificed in extremis and were subjected to necropsy. Further the animals were examined daily for evident signs of toxicity.

DETAILED CLINICAL OBSERVATION
Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. For the purpose of these examinations, the rats were transferred into a standard arena (50 x 37.5 x 25 cm). The following parameters were examined: abnormal behaviour during handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmia, appearance and consistency of faeces, urine and pupil size.

BODY WEIGHT
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION AND TEST ITEM INTAKE
Individual food consumption was determined weekly over a period of 1 day and calculated as mean food consumption in grams per rat and day.
The group mean daily intake of test item was calculated based upon individual values for body weight and mean food consumption per cage, according to following formula:
FCx X C /BWx
BWx = body weight on study day x (g)
FCx = mean daily food consumption on study day x (g)
C = test item concentration in the diet on study day x (mg/kg)

WATER CONSUMPTION
Daily visual check of the water bottles was done within the scope of general observations.

NEUROBEHAVIOURAL EXAMINATION, FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
The FOB was performed in all animals at the end of the administration period starting at about 10:00 h. It started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

- HOME CAGE OBSERVATION
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behaviour of the animals. Following parameters were considered: posture, tremor, convulsions, abnormal movements, impairment of gait, other findings.

- OPEN FIELD OBSERVATION
The animals were transferred into a standard arena (50 x 50 cm with sides of 25 cm high) and were observed for at least 2 minutes. Following parameters were examined: behaviour when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, faeces (number of faecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearing within two minutes.

- SENSORY MOTOR TESTS/REFLEX TESTS
The animals were removed from the open field and subjected to following sensorimotor or reflex tests:
1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response")
7. coordination of movements ("righting response")
8. behaviour during "handling"
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hind limbs
13. landing foot-splay test
14. other findings

MEASUREMENT OF MOTOR ACTIVITY (MA)
Motor activity measurements were carried out in all animals at the end of the administration period (same day as FOB).
The examinations were performed using the TSE Labmaster System [TSE Systems GmbH, Bad Homburg, Germany]. For this purpose, the animals were placed in new clean polycarbonate cages for the time of measurement; 18 beams were allocated per cage. The animals were put into the cages in a randomized order. The measurements started at about 14.00 h. The number of beam interrupts was counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

HEMATOLOGY AND CLINICAL CHEMISTRY
Blood samples were taken from the retro orbital venous plexus of the fasted animals in the morning (anaesthetizer was isoflurane); these samples were used for hematological and clinical-chemical examinations.
For hematology, following parameters were considered (all animals):
Hematocrit, hemoglobin (HB), mean cell hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), erythrocyte count (RBC), leucocytes count (WBC), differential blood cell count, reticulocytes, platelet count (PLAT), mean corpuscular volume (MCV). Clotting analyses also were done, and the prothrombin time was measured.
Following clinical chemical parameters were considered:
Sodium, potassium, glucose, chloride, calcium, inorganic phosphorus, blood urea, creatinine, total cholesterol, triglycerides, total protein, total bilirubin, albumin, globulins, magnesium, bile acids.
Following enzymes were considered:
Alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-Glutamyltransferase (GGT).

URINALYSIS
For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence. Volume, colour, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity and sediment were measured/examined.

Sacrifice and pathology:

Prior sacrifice, the anesthetized animals were weighed. They were then sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS
Following organs were weighed: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, spleen, brain, heart, thymus and thyroid glands. Both, the absolute and the relative organ weights were considered.

ORGAN/TISSUE FIXATION IN FORMALDEHYDE 4% SOLUTION
Following sacrifice, all organs were examined before and after removal. Any abnormality was noted.
Samples from following organs/tissues as well as gross lesions were collected and fixed in neutral 4% buffered formaldehyde for further histopathological examinations: all gross lesions, salivary glands (mandibular and sublingual glands), oesophagus, stomach (forestomach and glandular stomach), duodenum, jejunum and ileum, cecum, colon and rectum, liver, pancreas, brain, pituitary gland, sciatic nerve, spinal cord (cervical, thoracic and lumbar cords), eyes, adrenal glands, thyroid and parathyroid glands, trachea, lungs, pharynx, larynx, nose (nasal cavity), aorta, heart, bone marrow (femur), lymph nodes (mesenteric and axillary lymph nodes), spleen, thymus, kidneys, urinary bladder, gonads, oviducts, uterus and vagina, epididymides, prostate and seminal vesicle, female mammary gland, skin, skeletal muscle, sternum with marrow, femur with knee joint, extra orbital lacrimal glands.
It was specified that from the liver, each one slice of the Lobus dexter medialis and the Lobus sinister lateralis had been fixed in Carnoy’s solution and embedded in paraplast.

HISTOPATHOLOGY
The following organ samples of all animals per sex and group of the control and high dose group at test ending were processed for histological assessment (i.e. paraffin embedding, sectioning, haematoxylin and eosin staining):
Brain, pituitary, thyroid, thymus, trachea, lungs, heart, liver, spleen, kidneys, adrenals, testes, ovaries, uterus, vagina, cervix, epididymides, prostate, seminal vesicle, coagulating glands, Peyer's patches, stomach (forestomach and glandular stomach), duodenum/jejunum/ileum, cecum/colon/rectum, urinary bladder, mesenteric and axillary lymph nodes, sciatic nerve, femur bone marrow, eyes with optic nerve, skeletal muscle, sternum with marrow, all spinal cord. Gross lesions seen in all groups, also were processed for histopathology.
The haematoxylin-eosin stained slides were examined by light microscopy and assessed. A correlation between gross lesions and histopathological findings was performed.

Other examinations:

Particular attention was given to the immuno-relevant organs and tissues were evaluated according to the following parameters and criteria:
Thymus:
• Increased/decreased grade of cortico-medullar ratio (related only to area)
• Increase of starry sky cells
• Changes of cellular density in the cortex
• Changes of cellular density in the medulla
Spleen:
• Changes of the cellularity of PALS, lymphoid follicles, marginal zone, red pulp
• Altered cellular composition of follicles
• Altered number of germinal centers
Lymph nodes (mesenteric and axillary lymph nodes):
• Changes in the cellularity of follicles, interfollicular area, paracortical area, medulla
• Altered cellular composition of paracortex
• Altered number of germinal centers
• Hyperplasia of high endothelial venules
Peyer's patches (of the jejunum):
• Changes of the cellularity of follicles (including mantle zone and germinal centers)
• Changes of the cellularity of interfollicular area
Bone marrow:
• Changes of the cellularity
• Changes of the myeloid/erythroid ratio

Particular attention was further given to the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina.

Statistics:

The tests used for the statistical assessment of the results for the different parameters considered can be summarized as follows:
- body weight, body weight change: Comparison of each group with control group using DUNNETT's test (two-sided) for the hypothesis of equal means;
- Feces, rearing, grip strength forelimbs, grip strength hind limbs, footsplay test, motor activity, clinical pathology parameters, weight parameters at necropsy: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.

Details on results:

MORTALITY AND CLINICAL SYMPTOMS
No animal died prematurely. No treatment-related clinical symptoms were observed.

BODY WEIGHT [Tables 1A and 1B]
No treatment-related effects on body weight and/or body weight change were observed in males throughout the whole study.
Whereas the females of the low and mid dose group (1200 ppm and 4000 ppm) showed a significantly increase in body weight and body weight change at the end of the testing period (day 28), no such finding could be evidenced in the high dose group. Thus, because of the lack of a significant increase of these parameters in the high dose group, as well as the absence of a clear dose-dependency, the findings were not considered to be treatment-related.

FOOD AND WATER CONSUMPTION
Neither food nor water consumption was affected by the treatment in either sex throughout the whole study.

TEST ITEM INTAKE
The mean daily test item intake over the entire study period is summarized as follows:
In the low dose group (1200 ppm): the mean daily intake was 96 and 104.5 mg/kg bw/day for males and females, respectively.
In the mid dose group (4000 ppm): the mean daily intake was 330.5 and 332.5 mg/kg bw/day for males and females, respectively.
In the high dose group (12000 ppm): the mean daily intake was 977.5 and 1014.6 mg/kg bw/day for males and females, respectively.

FOB
The FOB of the treated animals was inconspicuous compared to control.

MOTOR ACTIVITY MEASUREMENT (MA)
Regarding the overall motor activity (summation of all intervals) as well as the single intervals, no treatment-related changes were observed.

HEMATOLOGY
In females of the mid dose group (4000 ppm) mean corpuscular volume (MCV) was found to be increased and in females of the low dose group (1200 ppm) mean corpuscular hemoglobin content (MCH) was below control values. However, both parameters were not changed dose-dependently and no other hematological parameter was changed. Therefore, these changes were considered to be incidental and not treatment-related, and thus, no treatment-related changes in the hematological parameters were reported.

CLINICAL CHEMISTRY
In females of the low and high dose groups (1200 and 12000 ppm) creatinine levels were measured to be increased compared to controls. However, the means were not changed dose-dependently and no other clinical chemistry parameters were affected. Therefore, these changes were considered to be incidental and not treatment-related, and thus, no treatment-related changes in the hematological parameters were reported.

URINE ANALYSIS
In males of the high dose group (12000 ppm) urinary specific gravity was found to be increased, and the urine volume was smaller but not statistically significantly. Since no further changes in urinary parameters were noticed, these findings were regarded as traducing an adaptation of the renal function to a decreased water intake and consequently as treatment-related, but not adverse effect.

NECROPSY
Both, the absolute and relative organ weights were not affected by the treatment when compared to control.
Gross lesions when occurring were single, incidental or spontaneous in nature. Thus, no treatment-related gross pathological findings were seen.
Histopathological findings when occurring were single or equally distributed between all groups including control, incidental or spontaneous in nature. Thus, no treatment-related histo-pathological findings were seen.

Dose descriptor:

NOAEL

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were seen in male and female rats treated up to the highest dose level of 12000 ppm.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to 12000 ppm

Dose descriptor:

NOAEL

Effect level:

1 014.6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: corresponding to 12000 ppm

Critical effects observed:

not specified

TABLE 1A & 1B:

TABLE 1A. Body weights [as mean in g and as % of development referring to control] of rats treated for 28 days with the test item in diet:
MALES [N=5/group]
Test group	Time	Day 0	Day 7	Day 14	Day 21	Day 28
Control [0 ppm]	mean	166.5±7.9	211.1±14.4	246.8±16.9	275.6±22.9	295.8±24.6
1200 ppm	mean	163.6±7.5	206.6±11.2	240.4±11.7	269.1±12.3	283.1±14.1
	% dev	-1.7	-2.2	-2.6	-2.4	-4.3
4000 ppm	mean	169.0±6.0	212.2±7.2	244.5±11.0	270.3±11.5	285.8±12.2
	% dev	1.5	0.5	-1.0	-1.9	-3.4
12000 ppm	mean	165.6±8.6	208.6±12.5	241.4±17.8	269.3±22.8	283.0±25.0
	% dev	-0.6	-1.2	-2.2	-2.3	-4.3
FEMALES [N=5/group]
Test group	Time	Day 0	Day 7	Day 14	Day 21	Day 28
Control [0 ppm]	mean	127.9±6.4	148.5±9.7	160.4±12.5	173.9±10.4	172.4±8.2
1200 ppm	mean	133.1±7.4	149.7±8.8	166.4±5.2	182.3±9.3	189.5±12.5*
	% dev	4.1	0.9	3.8	4.8	9.9
4000 ppm	mean	133.7±6.9	152.5±9.1	167.8±11.9	184.3±13.6	190.5±9.1*
	% dev	4.5	2.7	4.6	5.9	10.5
12000 ppm	mean	134.1±5.6	150.8±6.6	164.1±6.1	179.0±11.4	187.4±9.8
	% dev	4.8	1.5	2.3	2.9	8.7

 *, p<=0.05

TABLE 1B. Body weight changes [as mean in g and as % of development referring to control] of rats treated for 28 days with the test item in diet:
MALES [N=5/group]
Test group	Time	Day 0 - 7	Day 0 -14	Day 0 - 21	Day 0 - 28
Control [0 ppm]	mean	44.6±6.7	80.4±10.5	109.1±18.9	129.3±20.8
1200 ppm	mean	42.9±4.6	76.8±5.3	105.4±7.0	119.5±9.5
	% dev	-3.8	-4.4	-3.4	-7.6
4000 ppm	mean	43.2±2.6	75.4±6.7	101.3±7.5	116.8±9.7
	% dev	-3.2	-6.1	-7.2	-9.7
12000 ppm	mean	43.0±4.2	75.9±10.6	103.8±17.0	117.4±20.0
	% dev	-3.6	-5.6	-4.9	-9.2
FEMALES [N=5/group]
Test group	Time	Day 0 - 7	Day 0 - 14	Day 0 - 21	Day 0 - 28
Control [0 ppm]	mean	20.6±5.3	32.5±8.7	46.0±7.0	44.5±6.2
1200 ppm	mean	16.7±1.6	33.3±4.3	49.2±5.2	56.3±5.7**
	% dev	-18.9	2.4	6.9	26.7
4000 ppm	mean	18.8±3.1	34.2±5.7	50.6±7.0	56.8±2.6**
	% dev	-8.7	5.1	9.9	27.8
12000 ppm	mean	16.7±2.2	30.0±5.5	44.9±8.2	53.3±6.1
	% dev	-19.1	-7.8	-2.4	19.8

**, p<=0.01

Conclusions:

Under the conditions of the present study which was conducted according to the OECD TG 407 (2008), the NOAEL, was set at 12000 ppm for both male and female Wistar rats, corresponding respectively to 977.5 and 1014.6 mg/kg bw/day.

Reference 2

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

977.5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD guideline study conducted under GLP conditions.

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

For assessment of repeated dose toxicity, the test item 2,2'-Oxybisethanol, ethoxylated and propoxylated (>1 < 4.5 mol EO and >1 <4.5 mol PO) was administered to male and female Wistar rats via the diet at dose levels of 0, 1200, 4000 and 12000 ppm over a period of 4 weeks. Each group consisted of 5 animals per sex. The test was conducted according to the OECD 407 (2008) under GLP conditions.

The mean daily test item intake over the entire study period was as follows:

In the low dose group (1200 ppm): the mean daily intake was 96 and 104.5 mg/kg bw/day for males and females, respectively.

In the mid dose group (4000 ppm): the mean daily intake was 330.5 and 332.5 mg/kg bw/day for males and females, respectively.

In the high dose group (12000 ppm): the mean daily intake was 977.5 and 1014.6 mg/kg bw/day for males and females, respectively.

No mortality occurred. No adverse treatment-related effects could be reported referring to clinical symptoms, body weight and body weight gain, food and water consumption, FOB, motor activity, hematological and clinical-chemical parameters, organ weight, gross pathology and histopathology.

In males urinalysis revealed a statistically significant increase in the specific gravity and a decrease in of urinary volume which did not result in a statistical significance.

No crystals of calcium oxalates, pH shifts or other indications of renal toxicity were identified in any male dose group. The changes observed in male animals were therefore interpreted as adaptation of the renal function in response to a decreased water intake, and consequenttly as treatment related, but not adverse.

No effects were identified in the urinalysis of females.

Thus, under the conditions of the present study the no observed adverse effect level, NOAEL, was set at 12000 ppm for both male and female Wistar rats, corresponding respectively to 977.5 and 1014.6 mg/kg bw/day.

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 male and female Wistar rats, a NOAEL of 977.5 and 1014.6 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/d 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.