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

Repeated dose toxicity: oral

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-Oxybisethanol, ethoxylated and propoxylated (>1 < 4.5 mol EO and >1 <4.5 mol PO)

EC Number:

610-559-8

Molecular formula:

C4 H8 O3. ( C3 H6 O )a+c.(C2 H4 O)b+d ; 1 < a+c < 4.5; 1 < b+d < 4.5

IUPAC Name:

2,2'-Oxybisethanol, ethoxylated and propoxylated (>1 < 4.5 mol EO and >1 <4.5 mol PO)

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 2,2'-Oxybisethanol, ethoxylated and propoxylated (>1 < 4.5 mol EO and >1 <4.5 mol PO)
- Physical state: liquid, colorless, clear
- Analytical purity: Chemical-Spectroscopical analysis identified the test item as 2,2’-Oxybisethanol with approximately 2-3 EO units and 2-3 PO units (Analytical Study Report No. 11L00021, dated 25 May 2011, included in study report)
- Batch No.: T35/034/10
- Homogeneity: given (vis.)
- Stability: stable until 31 Dec 2012
- Storage condition of test material: room temperature

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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.

2

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

3

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.