1,1'-[oxybis(ethyleneoxy)]diethylene

Endpoint summary

Description of key information

The test item was administered to rats by intragastric intubation, daily, for twenty-eight consecutive days at dosage levels of 30, 100, 300 and 1000 mg/kg bw/day. The NOAEL was considered to be 1000 mg/kg bw/day in both the male and female rats (highest dose tested).

A testing proposal for a sub-chronic repeated dose toxicity study in rats (OECD 408) was submitted to ECHA for evaluation. As soon as the TPE is finalized the study will be conducted and the dossier will be updated after finalization of the report.

A study according OECD 422/GLP is available for the test substance. The test item was administered at 80, 250 or 800 mg/kg bw/day to Hannover Wistar rats for 28 days in males and 50-55 days in females. The NOAEL for systemic toxicity of the parental generation was 80 mg/kg bw/day for males based on clinical signs, body weight gain and food consumption effects on the high dose group and on kidney findings in male rats at the mid and high dose group. The NOAEL for systemic toxicity was 250 mg/kg bw/day for females based on clinical signs, body weight gain and food consumption effects. The NOAEL of 80 mg/kg bw/day was used for DNEL deviation.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

2021-05-31 to 2022-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

Hannover Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11/10 weeks old (males/females) at start of the treatment and 13/12 weeks old (males/females) at mating
- Weight at study initiation: (P) Males: 311-363 g; Females: 172-196 g
- Fasting period before study: no
- Housing: group-housed, up to 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation, delivery, lactation period, when they were paired or individually housed (with pups), respectively. Type II, III and/or IV polycarbonate cages
- Diet: ad libitum, ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance”
- Water: ad libitum, tap water from the municipal supply
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-25.0℃ (target range: 19-25°C)
- Humidity (%): 36-61% (target range: 30-70%)
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the selected vehicle (corn oil), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level. The formulations were stirred with magnetic stirrer during the preparation and shortly before the treatment and during the treatment. Prepared formulation was stored in a refrigerator (2°C to 8°C), protected from light for a maximum of 4 days prior to administration to animals according to stability assessment results of the analytical method development and method validation studies.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the results of a trial formulation, and the Dose Rage Finding (DRF) study and analytical method development study, corn oil was selected as vehicle for this study. Corn oil was considered as being an acceptable vehicle based on the scientific literature and practice of the Test Facility.
- Concentration in vehicle: 20, 62.5, 200 mg/mL
- Amount of vehicle: A constant volume of 4 mL/kg bw was administered to all animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The measured concentrations of the test item in the different formulations varied between 97% and 106% of the nominal concentrations. The results were considered to be acceptable. All test item formulations were shown to be homogeneous. The relative standard deviation (RSD) was below 10% in each case.

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period). Females were dosed for 14 days pre-mating, for up to 5 days mating period, then main animals were treated through gestation and up to and including the day before necropsy (13 days postpartum dosing). The day of birth (when parturition is complete) was defined as Day 0 postpartum.

Frequency of treatment:

daily

Dose / conc.:

80 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of a Dose Range Finding (DRF).In the DRF study (after a 14-day treatment period), at the 1000 mg/kg bw/day dose level in both sexes, transient clinical signs (decreased activity, increased salivation / salivation, piloerection, ataxia) were recorded for the majority of the animals, no similar findings were noted at 300 mg/kg bw/day (only salivation was observed). No test item related clearly adverse effect was seen on body weight or food consumption. No test item-related effects were seen on the examined clinical pathology parameters in the dosed groups. Liver weights adjusted for body weight were ~20% above control in both sexes at 1000 mg/kg bw/day, but there was no evidence at necropsy of any test item-related macroscopic findings in animals dosed at 300 or 1000 mg/kg bw/day. The high dose was set at 800 mg/kg bw/day; lower doses were spaced with a factor of approximately 3. Based on increased liver weights of ~20% after 14 days and taken into account the significantly longer administration period in the present study, the dose level of 1000 mg/kg bw/day was considered as probably too high. Therefore, a high dose of 800 mg/kg bw/day was selected for the present study. The other dose levels were selected to provide a dose-response relationship and a NOAEL at the low dose level.

- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality once per day in the pre-treatment period and twice daily in the treatment period (at the beginning and end of each working day). General (routine) clinical observations were made once a day, during the pre-treatment, treatment in the afternoon (pm). Routine clinical observation was performed twice daily from Day 7. No general clinical observations were made on the day of necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made at the start of the pre-exposure period on Day -7, and once before the first exposure on Day 0 (to allow for within-subject comparisons), then weekly (in the morning (am) or before treatment) and before necropsy. These observations were made outside the home cage in a standard arena, at similar times as practical. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self- mutilation, walking backwards) were also recorded.
Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. On Gestation Day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat). Furthermore, mated females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of 1 g weekly during the pre-exposure period, then on Day 0, and afterwards weekly, and at termination. Parent females were weighed at least on Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20, on PPD (Post-partum Day) 0 (i.e. within 24 hours after parturition), 4, 7, 10 and 13, and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Animal food consumption was determined by weighing the non-consumed diet with a precision of 1 g weekly (on body weight measurements day). Food consumption was measured more frequently during the gestation period (at least on GD 0, 3, 7, 10, 14, 17 and 20) and lactation period (at least on PPD 0, 4, 7, 10 and 13).

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Seven male and five female animals from each main group were selected for clinical pathology sampling and analysis at termination (on Day 28 in males, on PPD 14 in females).The following parameters were evaluated in animals selected:
RBC Red Blood Cell (erythrocyte) count, WBC White Blood Cell (leukocyte) count, Hgb Haemoglobin concentration, Hct Haematocrit, MCV Mean Corpuscular (erythrocyte) Volume, MCH Mean Corpuscular (erythrocyte) Haemoglobin, MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, RDW Red Cell (erythrocyte) Distribution Width, Plt Platelet (thrombocyte) count, MPV Mean Platelet Thrombocyte volume, RETIC % Reticulocyte count, NE % Neutrophil, LY % Lymphocyte, MO % Monocyte, BA % Basophil, EO % Eosinophil, LUC % Large Unstained Cells, APTT Activated Partial Thromboplastin Time, PT Prothrombin Time

CLINICAL CHEMISTRY: Yes
Seven male and five female animals from each main group were selected for clinical pathology sampling and analysis at termination (on Day 28 in males, on PPD 14 in females). The following parameters were evaluated in animals selected:
Glucose Blood sugar concentration, T-BIL Total Bilirubin concentration, Urea concentration, Cholesterol concentration, Creatinine concentration, Phosphorus concentration, Sodium concentration, Potassium concentration, Calcium concentration, Chloride concentration, Total Protein concentration, Albumin concentration, Alb/glob ratio, Aspartate Aminotransferase activity, Alanine Aminotransferase activity, Gamma-Glutamyl transferase activity, Alkaline Phosphatase activity, Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional Observational Battery and locomotor activity measurement was performed in the study. Randomly selected 5 males and 5 females per group:
The same animals were used as selected for clinical pathology. Assessment of any potential test item related neurotoxicity was performed during the last exposure week (for example, males on Day 23 am, females on PPD 6-8 am). Selected animals were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength. In order to avoid hypothermia of pups, dams were normally removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during locomotor activity measurement (SMART). A modified Irwin test was performed when sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted, and the general physical condition and behaviour of animals was tested. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. This was repeated 3 times for each animal. The distance between the two resulting ink spots of the hind limbs was measured. Fore/hind grip strength was measured using a grip strength meter, an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they release the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results are tabulated with individual and mean data. Locomotor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity. Locomotor activity was monitored by placing each animal individually into a
50 x 50 cm open-field for 1-hour observation time, DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments was presented graphically with the intention of showing plateau activity in controls and comparing the treatment groups.

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE
Animals were euthanized at termination. After blood sampling, gross necropsy was performed on each animal (with special attention to reproductive system and any expected target tissues). Weight of selected organs were measured, and selected organs and tissues were retained.

All animals including the selected animals for blood sampling were fasted (overnight period of food deprivation, in case of females this happened after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
For thyroid hormone analysis, blood samples were taken by venepuncture (using vena sublingualis in case of adult animals) or decapitation (in case of pups) as follows:
- from all dams on PPD 14
- from all non-pregnant adult females on the day of termination,
- from all adult males on the day of termination
The timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight and weight of the following organs of all adult animals was determined:
- with a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and
thymus,
- with a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids.
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight isreported for each animal adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.

On completion of the macroscopic examination the following tissues and organs were retained from all adult animals. The eyes with the optic nerve, testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.

Gross findings, Lungs with bronchi, Skeletal muscle (quadriceps), Adrenal gland, Lymph node, Small intestine, Ovary, Spinal cord, *Aorta, Oviduct, Spleen, Brain, *Pancreas, Sternum with marrow, Cervix, Larynx, *Pharynx, Epididymis, *Pituitary, Stomach, Eye with the optic nerve, Prostate, Testis, *Oesophagus, *Salivary gland (including mandibular, sublingual and parotid glands), Thymus, *Mammary gland, *Nose, *Femur with marrow (with knee joint), Thyroid with parathyroid gland, Heart, Tongue, Kidney, Sciatic nerve, Trachea, Large intestine, Seminal vesicle with coagulating gland, Urinary bladder, *Extraorbital lachrymal gland, Uterus, Harderian gland, Skin, subcutis with mammary gland (inguinal), Vagina

*= no histopathological examination

In case microscopic examination was needed for a tissue or organ, the retained tissues and organs required for histopathology were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylineosin/phloxine and examined by light microscope. For the adult animals, detailed histological examination was performed as follows:
- on the selected list of retained tissues and organs (as above) in the Control and High dose groups: selected 5 animals/sex/group (the same animals were used for clinical
pathology)
- all macroscopic findings (abnormalities), except of minor order from all animals
- on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina
for females) of all animals of the Control and High dose groups, and of all males that failed to sire and all females that failed to deliver healthy pups.

Additional immunohistological staining was performed on all High dose male (12 animals) and all Control male (12 animals) kidneys for alpha 2u globulin and additional histological examination was performed on kidneys of parental males and the stomach (non-glandular) of all remaining parental males and females.

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention was paid to the organ weight, appearance and histopathology of immunesystem tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.

Statistics:

In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data.
In case of the SAS 9.3 software package the following decision tree was applied for statistical evaluation of continuous numeric data. The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of (co)variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences. If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid, and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test. For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control. For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item related clinical signs were observed in the Control, Low and Mid dose male or female animals.
Slightly decreased activity was observed for 8/12 High dose males from Day 7 to Day 27 and for 8/12 High dose females on Day 16, 18, 27, 33, 40 and 46. The longevity of the symptom was 8 days.
Slight ataxia was observed for 1/12 High dose males on Day 18 and for 5/12 High dose females from Day 16 to 18. The longevity of the symptom was 1 to 3 days.
Slightly to moderately increased salivation was observed for 12/12 High dose males from Day 14 to Day 27 and 12/12 High dose females from Day 15 to Day 54. The longevity of the symptoms was 35 days.
Piloerection was observed for 3/12 High dose males from Day 4 to Day 27. The longevity of the symptom was 3 days.
Recumbency was observed for 4/12 High dose males from Day 7 to Day 21 and 3/12 High dose females from Day 47 to Day 51. The longevity of the symptoms was 4 days.
Red discharge (nose) was observed for 1/12 High dose male on Day 19.
Red coloured urine was observed for 1/12 High dose female on Day 16.
Alopecia was observed for 1/12 Low dose female from Day 34 to Day 50 and for 1/12 Mid dose female from Day 49 to Day 53. The longevity of the symptom was 17 days.
Thin fur was observed for 2/12 Control females from Day 35 to Day 52 or 53, for 3/12 Low dose females from Day 29 to 50 or 51, on Day 53 and for 1/12 Mid dose female on Day 51. The longevity of the symptoms was 1 or 21-22 days.
Decreased activity, ataxia, increased salivation, piloerection, recumbency, red discharge and coloured urine were considered as test item related effects, the others were incidental, not related to the treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality or morbidity was observed in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant test item related effect on body weight or body weight gain was detected in the study for males. In males, the body weights of the High dose appeared to show lower growth throughout the study (weight growth was ~42% lower than control for D0-27), with the Day 27 body weight ~5% lower than control. Although these differences were not statistically significant, the pattern of growth relative to the pre-treatment period indicates a slight treatment related effect.
Statistically significantly lower (p<0.05) body weight, by about 5%, was observed during the lactation period in the High dose female group, the difference to control is small, but may be a treatment related effect.Statistically significantly decreased body weight gain was observed in the High dose female group between Day7-14 (p<0.05) and Day0-14 (p<0.01), with ~44% lower weight gain to day 14 (similar to the High dose males). The body weight profile of the test item treated animals was comparable with control for the Low and Mid groups, with slightly lower growth in males and females at the High dose, with minor differences occasionally.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effect on food consumption was seen in the first two weeks of the treatment in the High dose group (males and females).
Statistically significantly decreased food consumption was observed in the High dose male group (p<0.01 and p<0.05) throughout the treatment period. Data for Day 0-14 period was outside of the historical control range and together with the body weight gain data, it was considered as test item related effect.Statistically significantly decreased food consumption was observed in the High dose female
group during the first two weeks (decreased by -12.6% compared to the Control) (p<0.01 and p<0.05) and during lactation period (decreased by -17.9% compared to the Control) (p<0.01). No such effect was observed during gestation period. The values for High dose females in the first two weeks were out of the historical control range and together with the body weight gain data it was considered as test item related effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes in the male and female dosed group.
Statistically significantly increased neutrophil relative (p<0.01) was observed in the High dose male group when compared to control. Statistically significantly decreased reticulocyte relative (p<0.05) and lymphocyte relative (p<0.01) was observed in the High dose male group when compared to control. The majority of the observed values were within the historical control range, thus these differences were considered as animal variability, not being a test item related effect. No statistically significant and biologically relevant changes were recorded for any other haematology parameters in the test item treated males and females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were test item related changes on the serum chemistry in the High dose female group that could be ascribed to the test item administration.
Statistically significant increase (p<0.05) in urea concentration was observed in High dose males compared to control. Two of the animals were well above the historical control range therefore, the observed differences were considered as test item related, but in isolation it may not be an adverse effect. Statistically significantly increased calcium level (p<0.05) which appears to be treatment related (and may correspond with kidney histology) increased and bile acid (p<0.01) level was observed in the High dose female group, where the mean value was in the historical control range, but the tendency for high values suggests a treatment related effect.Alkaline phosphatase activity and alanine aminotransferase (ALT/GPT) activity was statistically significantly increased in the High dose female group. The observed data were not considered to be excessively high; the statistical differences are of equivocal relationship with treatment.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test item related effects on the T4 and TSH hormone levels and the thyroid gland weight for any of the adult males. The TSH hormone level was measured at the Test Site and later at the Test Facility also to strengthen the outcome of the measurements in case of the male animals. The measurement of the thyroid hormone levels in the adult females, and histology of pup thyroids were not performed as it was not deemed necessary. Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted in the Irwin test or during the assessment of grip strength and landing foot splay.
Statistically significantly decreased locomotor activity total distance travelled, was observed for the High dose male group for the first 20 minutes (p<0.01 and p<0.05) compared to the Control, but in the plateau phase (20-60 minutes) the data was not different to the other groups. The other dose groups of males and females had a normal locomotor activity. In all cases, the initial activity was relatively high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. All treated groups had a general profile of activity the same as historical control data. The High dose males had initially lower locomotor activity, but this was no seen at the regular detail observation times in the study, or at the FOB, and females were clearly unaffected, furthermore the profile of the activity over time was fully consistent with normal animals. It is considered doubtful that the statistical differences were a real treatment related effect.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males: Treatment-related effects were observed in the kidney weights in High dose males. The absolute and relative (to body and brain) weights of the kidney increased statistically significantly (by 42.9%, 53.7% and 44.4%, respectively) in the High dose group compared to Controls, with histological relevance.
The absolute and relative (to body and brain) weights of the thymus decreased statistically significantly (by 25.3%, 21.1% and 25.0%, respectively) in the High dose group compared to Controls without histological relevance, and data were within the historical control range, these statistical differences were not considered as an adverse effect of the test item. The relative to body weights of the testes increased statistically significantly (by 10.8%) in the High dose group compared to Controls but the absolute weights were no affected and there was no histological relevance. The data were also within the historical control range; the statistical difference was not considered as a significant test item effect on the testis.
Females: There were statistically significant differences among groups in the weights of a few organs (kidneys, liver and spleen) either at absolute and/or related to body weight measured when compared to Controls. However, in the absence of any macroscopic or microscopic correlates and inconsistent direction or small magnitude, these weight changes were considered as not related to test item administration.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item related changes were observed in the kidney in males. Small thymus was observed in 2/12 High dose males and 1/11 High dose females which is unrelated to treatment.All other observed changes were considered incidental or a common background.

Necropsy examination did not show any test item related change in the non-pregnant females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were observed in the kidney in males, and stomach. Immunohistochemical staining for alpha 2 u globulin revealed comparable incidence between Control and High dose male kidneys.

Minimal to mild diffuse squamous cell hyperplasia of the non-glandular stomach was observed in 5/12 High dose males and 4/11 High dose females. Minimal squamous hyperplasia observed in single males from Low and Mid dose males and a 3/11 Mid dose females may be considered within the common background.

Macroscopically, small thymus was observed in 2/12 High dose males and 1/11 High dose females. Microscopically, minimal to mild reduced cellularity of the cortex was observed in the thymus in 1/6 High dose males and 3/5 High dose females. Considering the low incidence of macroscopic and microscopic findings at low severity (minimal mostly) in the thymus and organ weight changes only in high dose males, these changes are considered incidental and not related to test item administration. All other changes seen in Control and/or treated animals, were without meaningful differences in severity and incidence, therefore were regarded as incidental, procedural or a common background.

No microscopic changes were observed in the non-pregnant females.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study planned

Study period:

After approval by ECHA

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE: 1,1'-[oxybis(ethyleneoxy)]diethylene
- Name of the substance on which testing is proposed to be carried out: 1,1'-[oxybis(ethyleneoxy)]diethylene (CAS 764-99-8, EC 212-133-3)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
- Available GLP studies: There are no GLP studies available covering sub-chronic repeated dose toxicity information requirements.
- Available non-GLP studies: There are no non-GLP studies available covering sub-chronic repeated dose toxicity information requirements.
- Historical human/control data: There are no historical human data available on repeated dose toxicity for the substance.
- (Q)SAR: At present there is no valid (Q)SAR model available to address repeated dose toxicity (ECHA Guidance in Information Requirements and Chemical Safety Assessment Chaper R 7a: Endpoint specific guidance).
- In vitro methods: At present there are no valid in vitro methods available to address repeated dose toxicity (ECHA Guidance in Information Requirements and Chemical Safety Assessment Chaper R 7a: Endpoint specific guidance).
- Weight of evidence: There are no data available which are sufficient for weight of evidence approach.
- Grouping and read-across: No substances or a category of substances are known which apply for read-across addressing repeated dose toxicity study.
- Substance-tailored exposure driven testing: not applicable
- Approaches in addition to above: not applicable
- Other reasons: not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
No sub-chronic or chronic repeated dose toxicity study is available for the substance. According to Column 2 Annex IX of REACH Regulation a sub-chronic repeated dose toxicity study does not need to be conducted if:
- a reliable short-term toxicity study (28-days) is available showing severe toxicity effects
- a reliable chronic toxicity study is available
- the substance is unreactive, insoluble and not inhalable and there is no evidence of absorption and no evidence of toxicity in a 28-day “limit test”, particularly if such a pattern is coupled with limited human exposure.
All points outlined above do not apply for the substance. Thus, in order to fulfill information requirements of REACH Regulation for substances manufactured or imported in quantities of 100 tpa or more, a sub-chronic toxicity study (90-days) is proposed.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: A oral repeated dose toxicity study according to OECD 408 in the rat is proposed.

Qualifier:

according to guideline

Guideline:

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

Species:

rat

Route of administration:

oral: gavage

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

80 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD 422/GLP

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

OECD 407

The test item was administered to rats by intragastric intubation, daily, for twenty-eight consecutive days at dosage levels of 30, 100, 300 and 1000 mg/kg bw/day (Charles River Laboratories, 2007). Microscopic observations attributed to the test article were limited to minimal to mild epithelial hyperplasia and hyperkeratosis in the non glandular region of the stomach of both males and females treated with 1000 mg/kg bw/day and one female treated with 300 mg/kg bw/day. There was no other toxicity observed in the male and female rats as high as 1000 mg/kg bw/day. The stomach findings were considered of minimal toxicological significance and possibly an indication of local irritation rather than systemic toxicity. Therefore the no-observable-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg bw/day in both the male and female rats (highest dose tested).

DRF for OECD 422

In a Dose Range Finding (DRF) study for a subsequent OECD 422 study, the substance was formulated in corn oil and administered by oral gavage to Han-Wistar rats for 14 consecutive days at dose levels of 300 and 1000 mg/kg bw/day (BASF SE, 2021) The dose volume was 4 mL/kg body weight. There was no mortality in the study. Salivation and post-dosing decreased activity were considered to be High dose effects. Additionally, piloerection and ataxia were observed in individual occasions. High dose males tended to have lower weight gain and food intake, but without statistical significance. No test item-related adverse effects were seen on the examined clinical pathology parameters in the dosed groups. The High dose in both sexes tended to have adjusted liver weights of ~20% above control but no test-item related macroscopic findings were seen at necropsy. 

OECD 422

A study according OECD 422/GLP was performed. The test item was administered at 80, 250 or 800 mg/kg bw/day to Hannover Wistar rats for 28 days in males and 50-55 days in females. The dose levels were selected based on the results of a Dose Range Finding (DRF) study.

Parameters measured during the study included twice a day mortality checking, daily routine and weekly detailed observation of clinical signs, weekly body weight and food consumption measurements and clinical pathology evaluation (including hematology, coagulation, clinical chemistry and urinalysis). Neurological assessment (Functional Observation Battery (FOB) including measurements of the landing foot splay, grip strength, Irwin test as well as locomotor activity measurement) was performed during the last week of the treatment for each sex. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination (Day 28 for males, PPD (Post-partum Day) 14 for females), necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals or F1 animals. T4 and TSH levels in the PND (Post-natal Day) 13 pups and parental males were also determined. For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 animals/sex in the Control and High dose groups, reproductive organs of all animals of Control and High dose groups, all those male / females mating pairs where no liveborn pups were achieved. Dosing formulations were analyzed for concentration and/or homogeneity on three occasions during the study. Overall, the formulations were considered adequate for the study.

Decreased activity, ataxia, increased salivation, piloerection, recumbency, red discharge and colored urine were observed in some High dose male or female animals and were considered as test item related effects. Slightly reduced growth of adult High dose males (D0-27) and females (D0-14), and female weights in the lactation phase were considered to be treatment related. Test item related adverse effect on food consumption was observed in the first two weeks of the treatment in the High dose group of both sexes, correlating with the body weight data. There were no changes in animal behavior, general physical condition or in the reactions to different type of stimuli related to test item treatment when compared to control. There were no adverse test item related changes in the hematology parameters in the male and female dosed group. Test item related increase in the alkaline phosphatase activity and alanine aminotransferase activity and possibly increased plasma calcium, was observed in the High dose female group and increased urea in the High dose male group. No test item effect on estrus cycle of parental females was noted. No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding was recorded for F1 pups at necropsy. The High dose pups were slightly smaller and had slightly lower growth rates than controls, but this was most probably related to maternal toxicity and was not attributed to a direct effect on the pups. Histological kidney changes in the High dose males were characterized by tubular basophilia, tubular dilatation and presence of crystals in the tubular lumen. It is considered that either the test item or its metabolites are excreted through the kidney which contribute to the partial blockage of the tubule and contributes to other spectrum of changes which was collectively diagnosed as retrograde nephropathy. The tubular basophilia and dilatation without the presence of crystals in the tubules were recorded as independent terminologies, even though they are considered as part of the spectrum of changes contributing to retrograde nephropathy. The microscopic changes observed in the male kidneys correlated with organ weight changes and macroscopic observations. Immunohistochemical staining for alpha 2 u globulin revealed comparable incidences between Control and High dose male kidneys. Therefore, although the accumulation of proteinaceous droplets in the kidney tubules appears to be specific for male rats, the mechanism differs from the classic mechanism involving alpha 2 u globulin. However, these changes are considered potentially as adverse since the relevance for humans cannot be completely ruled out. Non-glandular gastric squamous cell hyperplasia (minimal to mild) was observed in both sexes at the High dose. Kidney findings were also present in males of the Mid dose group, but to a lesser extent.

Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.

The NOAEL for systemic toxicity of the parental generation: 80 mg/kg bw/day for males based on clinical signs, body weight gain and food consumption effects on the high dose group and on kidney findings in male rats at the mid and high dose group.

The NOAEL for systemic toxicity was 250 mg/kg bw/day for females (based on clinical signs, body weight gain and food consumption effects).

The NOAEL for reproductive effects of the parental generation: 800 mg/kg bw/day (based on no findings). The NOAEL for pups’ (F1 generation) development and survival: 800 mg/kg bw/day (based on no specific findings that were not attributed to maternal toxicity).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item is not classified and labelled for repeated toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.