Registration Dossier

Administrative data

Description of key information

oral
Rat OECD 422: NOAEL systemic toxicity = 100 mg/kg bw due to decreased body weight, food consumption, increased liver weights and microscopic liver changes in the higher dose (GLP, RTC/ Rohm & Haas 2004)
Rat OECD 408: NOAEL systemic toxicity = 80 mg/kg due to effects in liver (adaptive) and kidney, severe clincal signs and deaths at higher doseages (ERBC 2020)

dermal
no data available
inhalation
no data available

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
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sep 2019 - Sep 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL: BASF SE
Identity Bis(2-(2-butoxyethoxy)ethoxy)methane
CAS no. 143-29-3
Batch no. 00975536W0
Retest date 24 December 2020
Storage conditions Room Temperature
ERBC No. 16564

Analysis was performed in a separate study in order to validate the analytical method
and the preparation procedure in the concentration range of 1-100mg/mL (RTC Study no.
94840)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Before treatment, analysis was performed to confirmthat the proposed formulation
procedure was acceptable and that the stability of the preparations was satisfactory. The
preparations, in the concentration range of 8-80mg/mL,were stable for 24 hours at room
temperature and for 8 days at +4°C.
Samples of the preparations made inWeeks 1 and 13 of the current study were analysed
to check the homogeneity and concentration. Results of the analyses were within the
acceptability limits stated in ERBC SOPs for concentration suspension (85-115%) and
homogeneity (CV% < 10).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was administered orally by gavage at a dose volume of 10mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered
to each animal on the basis of the most recently recorded body weight and the
volume administered was recorded for each animal.
The vehicle was an aqueous solution of carboxymethylcellulose (CMC 0.5% w/v).
Species:
rat
Strain:
Sprague-Dawley
Remarks:
from Envigo RMS srl, San Pietro al Natisone (UD), Italy and supplied by Envigo Netherlands.
Details on species / strain selection:
A total of 130 Hsd: Sprague Dawley SD rats (65 males and 65 females), 27-29 days old and
with body weight of approximately 75-99 g, were ordered from Envigo RMS srl, San Pietro
al Natisone (UD), Italy and supplied by Envigo Netherlands.
After arrival the weight range for each sex was determined and found to be 94.4-123.1 g for
males and 93.5-106.3 g for females.
Sex:
male/female
Details on test animals and environmental conditions:
The animalswere housed in a limited access rodent facility. Animal roomcontrolswere set to
maintain temperature and relative humidity at 22°C±2°Cand 55%±15%, respectively; actual
conditions were monitored, recorded and the records retained. No relevant deviations
from these ranges were recorded during the study. There were approximately 15 to 20 air
changes per hour and the rooms were lit by artificial light for 12 hours each day.
The animals were housed up to 5 of one sex to a cage, in polisulfone solid bottomed cages
as indicated in the relevant SOP. Nesting material was provided inside suitable bedding
bags and changed at least twice a week.
Drinking water was supplied ad libitum to each cage via water bottles.
A commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei 4,
20019 SettimoMilanese (MI), Italy) was offered ad libitum throughout the study
Dated and signed records of activities relating to the day to day running and maintenance
of the study in the animal house were recorded.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was suspended in the vehicle. The preparationswere made
daily or weekly (concentrations of 8, 24 and 80mg/mL). Concentrations were calculated
and expressed in terms of test item as supplied.

VEHICLE
The vehicle was an aqueous solution of carboxymethylcellulose (CMC 0.5% w/v).
The test item was administered orally by gavage at a dose volume of 10mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered
to each animal on the basis of the most recently recorded body weight and the
volume administered was recorded for each animal.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate the analytical method
and the preparation procedure in the concentration range of 1-100mg/mL (RTC Study no.
94840). Before treatment, analysis was performed to confirmthat the proposed formulation
procedure was acceptable and that the stability of the preparations was satisfactory. The
preparations, in the concentration range of 8-80mg/mL,were stable for 24 hours at room
temperature and for 8 days at +4°C.
Samples of the preparations made inWeeks 1 and 13 of the current study were analysed
to check the homogeneity and concentration. Results of the analyses were within the
acceptability limits stated in ERBC SOPs for concentration suspension (85-115%) and
homogeneity (CV% < 10)
Duration of treatment / exposure:
13w
Frequency of treatment:
daily
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
240 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Each group comprised 10 male and 10 female rats sacrificed after 13 weeks of treatment
(main phase) and 5 additional animals per sex sacrificed after 4 weeks of recovery.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A GLP conform repeated dose/ reproductive toxicity study was performed with TP-90B RUBBER CHEMICAL according to OECD 422 (RTC/ Rohm & Haas 2004). Groups of 10 Sprague-Dawley rats per sex received TP-90B Rubber Chemical in 0.5% aqueous carboxymethylcellulose at doses of 10, 100 and 800 mg/kg bw/d by gavage. Males were treated for 2 weeks prior to pairing, through pairing until the day before sacrifice. Females were treated for 2 weeks prior to pairing, through the mating and gestation periods up to day 3 of lactation. Dams and offspring were sacrificed on Day 4
The NOAEL of this study was 100 mg/kg bw/d since increases in absolute and relative kidney weights noted in the mid-dose male group only were not interpreted as a clear adverse health effect as this effect was not observed in the high-dose group. Based on this low effects in this study compareable dose selections are made for the OECD 408 but sattelites are added due maybe adative Liver effects.
- Rationale for animal assignment (if not random): The rats were allocated to groups by computerised stratified randomisation
to give approximately equal initial group mean body weights
- Rationale for selecting satellite groups: within previous OECD 422 study maybe adaptive liver effects are investigated, therefore it was decided to add recovery groups to investigate this effect.
- Post-exposure recovery period in satellite groups: 4 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Mortality:
Throughout the study, all animals were checked early in each working day and again in
the afternoon. At weekends and Public Holiday a similar procedure was followed except
that the final check was carried out at approximately mid-day. This allowed post mortem
examinations to be carried out during the working period of that day.

DETAILED CLINICAL OBSERVATIONS: Yes
All clinical signs were recorded for individual animals. Once before commencement of
treatment, twice daily during the dosing period and once daily during recovery, each animal
was observed and any clinical signs recorded. Observations were performed at the same
time interval each day (0.5 - 1 hour and 2 - 2.5 hours post-dose). The intervals were selected
taking into consideration the presence of post-dose reactions.
Once before commencement of treatment and at least once per week during the study
from the start of treatment, each animal was given a detailed clinical examination. Each
animal was observed in an open arena. The test included observation of changes in gait
and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies
or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation,
piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes,
occurrences of secretions and excretions were also recorded.
Once duringWeeks 12 of treatment and once duringWeek 4 of recovery an evaluation of
sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive
stimuli) and an assessment of grip strength was also performed.

BODY WEIGHT: Yes
Each animal was weighed on the day of allocation to treatment group, on the day that
treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food consumed by each cage of rats was recorded at weekly intervals starting
from treatment. The group mean daily intake per rat was calculated.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
Both eyes of all animals were examined prior to the commencement of treatment by means
of an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropicamide
(Visumidriatic®, Visufarma, Rome, Italy). The eyes of all animals from high dose
and control groups were re-examined duringWeek 13 of treatment.

HAEMATOLOGY: Yes
During the last week of treatment under conditions of food and
water deprivation blood samples were collected under isoflurane anaesthesia from the
retro-orbital sinus of the same animals for thyroid hormone determination and haematology
and clinical chemistry investigations.During the necropsy procedure, blood samples
were collected under isoflurane anaesthesia from the abdominal vena cava for coagulation
determination. Further blood samples were taken under identical conditions at the end of the recovery
period.
The blood samples collected were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests
– No anticoagulant for hormone determination

Parameters checked:
- Haematocrit
– Haemoglobin
– Red blood cell count
– Reticulocyte count
– Mean red blood cell volume
– Mean corpuscular haemoglobin
– Mean corpuscular haemoglobin concentration
– White blood cell count
– Differential leucocyte count
· Neutrophils
· Lymphocytes
· Eosinophils
· Basophils
· Monocytes
· Large unstained cells
– Platelets
These parameters were analysed by Siemens Advia 120.
Coagulation
– Prothrombin time
This parameter was analysed by Instrumentation Laboratory ACL Elite PRO.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see Haematology

Parameters checked:
– Alkaline phosphatase
– Alanine aminotransferase
– Aspartate aminotransferase
– Gamma-glutamyltransferase
– Urea
– Creatinine
– Bile acids
– Glucose
– Triglycerides
– Inorganic phosphorus
– Total bilirubin
– Total cholesterol
– HDL
– LDL
– Total protein
– Albumin
– Globulin
– A/G Ratio
– Sodium
– Potassium
– Calcium
– Chloride
These parameters were analysed by Siemens Advia 1800.

URINALYSIS: Yes
During the last week of treatment, individual overnight urine samples were collected from
all male and female animals from each main phase group under conditions of food and
water deprivation

Parameters checked:
Volume (manually recorded)
– Appearance
– Specific gravity
– pH
– Protein
– Glucose
– Ketones
– Bilirubin
– Urobilinogen
– Blood
These parameters were analysed by Aution Eleven AE 4020 / Aution Sticks 10EA or ATAGO
Refractometer (for Specific gravity), according to internal procedures.
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes,
was examined microscopically for:
– Epithelial cells
– Leucocytes
– Erythrocytes
– Crystals
– Spermatozoa and precursors
– Other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes
The motor activity (MA) of all animals was measured once duringWeek 12 or 13 of treatment
and once duringWeek 4 of recovery by an automated activity recording. Measurements
were performed using a computer generated random order.

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Thyroid hormone determination (T3, T4 and TSH)
Samples (approximately 0.8 mL) were transferred into tubes containing no anticoagulant
and centrifuged at room temperature. The serum obtained was divided in two aliquots
and stored at -80°C pending analysis. Samples were assayed to determine the serum levels
of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating
hormone (TSH) by a multiplex assay, using LuminexMagpix system and the MILLIPLEX
MAP Rat ThyroidMagnetic Bead Panel kit (MerkMillipore, cat. No. RTHYMAG-30K).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The clinical history of the animals was studied and a detailed post mortem examination was conducted
From all animals completing the scheduled test period, the organs indicated in section were dissected free of fat and weighed

HISTOPATHOLOGY: Yes (see table)

Abnormalities
Adrenal glands
Aorta
Bone marrow (from sternum)
Brain (cerebrum, cerebellum,medulla/pons)
Caecum
Coagulating glands1
Colon
Duodenum
Epididymides
Eyes
Femur with joint
Harderian glands
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Mammary area (Males and Females)
Oesophagus
Ovaries
Oviducts
Pancreas
Parathyroid glands
Pituitary gland
Prostate gland
Rectum
Salivary glands
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal column
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach
Testes
Thymus (where present)
Thyroid gland
Trachea
Urinary bladder
Uterus – cervix
Vagina

Other examinations:
Oestrous cycle
At the end of the study, just prior to necropsy, vaginal smears were taken from all surviving
female animals, and the oestrous cycle phase recorded.
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables
the significance of the differences amongst groups was assessed by analysis of variance.
Differences between each treated group and the control group were assessed by Dunnett’s
test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s
test before Dunnett’s test. If the data were found to be inhomogeneous aModified t test
(Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the
actual values in the computer without rounding off. Statistical analysis of histopathological
finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Dosing phase
Treatment-related clinical signs included salivation, that was noted in all males and majority
of the females dosed at 800 mg/kg/day (Group 4) and most animals dosed at 240 mg/kg/day
(Group 3), ataxia, prone position and lethargy, seen in the majority of males and females
dosed at 800 mg/kg/day and with a lower incidence, in those receiving 240 mg/kg/day. In
addition, convulsions and/or tremors were occasionally observed during the treatment
period in 3 out of 10 males dosed at 800 mg/kg/day and several females of the same
treatment group and, with a lower incidence, in those treated at 240 mg/kg/day.
Hunched posture, piloerection, hairloss and decreased activity were also occasionally
observed in males and females dosed at 800 and 240 mg/kg/day.
No significant clinical signs were observed in animals dosed at 80 mg/kg/day (Group 2).
Recovery phase
Treatment- related clinical signs were no longer apparent during the recovery phase and
clinical signs were limited to moderate staining of the muzzle in one animal of Group 2 and
damaged eye in 1 or 2 animals of all groups.

Weekly detailed clinical signs
Dosing phase
Slight but statistically significant reduction in rearing was noted in treated animals during
the treatment period. The rearing was reduced in males of Groups 3 and/or 4 on Days 4, 18,
46, 53, 74, while females of Group 4 showed statistically significant reduction in rearing on
Days 5, 12 and 26. Statistically significant increase in rearing was recorded in male animals
of Group 3 on Day 81 and in females of Groups 2 and 3 on Day 5 of dosing period. No
toxicological significance was ascribed to other and inconsistent fluctuations observed
during the study.
Recovery phase
Slight and statistically significant decrease in rearing was recorded in male animals of Group
3, and females of Groups 2 and 3 on Day 25 of the recovery phase. This change is considered
of no toxicological relevance, since no other significant alterations were noted during the
recovery period.
Mortality:
mortality observed, treatment-related
Description (incidence):
On Day 71 of the dosing phase, several cases of premature death occurred in the high dose
group (Group 4).
The following animals of the treatment group were found dead:
– One male animal. Prone position was observed before the death.
– Five females of the treatment group, prone position,dyspnoea and lethargy were observed before the death.
In the same day of treatment, due to the severe clinical signs observed (dyspnoea, lethargy,
prone position, closed eyes, moribund status), 8 males and 9 females of the Group 4 were
sacrificed for humane reasons.
The macroscopic observation performed during the necropsy did not reveal any relevant
changes to determine the causes of illness or death of these animals.

One female of the low dose group, was found dead on Day 24 of the recovery period. No clinical signs were observed before the death. The macroscopic observation performed during the necropsy (red fluid in the skull, red colour in the meninges and in the muzzle, broken upper incisors) suggested that the cause of the death could be related to an incidental trauma.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences between treated and control animals were recorded during the dosing and
recovery period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slight, but statistically significant increase in food consuption, ranging from +6% to 13%,
was recorded in male animals from all treatment group (with no dose-relation), from Day
22 to Day 64 of the dosing period, when compared to controls. For female animals a slight
and statistically increase in food consuption was observed sporadically and with no doserelation
during the treatment period. In particular, On Day 22 this increment was recorded
for females of group 2 (+9%), on Day 57 in females of Group 4 (+7%) and on Day 64 for
those of Groups 3 and 4 (+10% and +12%), respectively.
These differences were no longer observed during the recovery period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Before the start of treatment, animals showing no ocular abnormality at the ophthalmoscopic
examination were selected for the study. Both eyes of control and surviving high
dose animals were re-examined duringWeek 13 of treatment (on Study Day 86). No ocular
findings were detected.
Haematological findings:
no effects observed
Description (incidence and severity):
Dosing phase
One male animal treated at 800mg/kg/day (no. A3671104) showed relevant neutrophilia.
Compared with mean control data, the increment was 9.5 fold. Due to the minimal incidence, this finding was considered to be unrelated to treatment.
Mean corpuscular volume was statistically significantly higher than controls in males dosed at 800mg/kg/day. Due to the minimal severity (4% above controls) and the absence of other related changes, this finding was considered to be not adverse.
Recovery phase
No differences between control and treated animals were observed.

Coagulation
Dosing Phase
Prothrombin time was increased in some males dosed at 800mg/kg/day (7% above controls).
Due to the minimal severity and the absence of other related changes, this finding was considered to be not adverse.
Recovery phase
No changes were recorded.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Dosing phase
Cholesterol was increased in animals of both sexes dosed at 240 and 800mg/kg/day. HDL and LDL cholesterol were both affected. Mean group data were 24% and 28% above controls in males, respectively, and 44% and 53% above controls in females, respectively. In males, this finding was recorded in two animals/group only for Group 3 and for Group 4 while in females it was recorded in 4 animals dosed at 240mg/kg/day and in one female receiving
800mg/kg/day.
Other statistically significant differences from control were recorded in treated males, such as: increase of calcium in males dosed at 240 and 800mg/kg/day (5% and 7%, respectively), decrease of bilirubin and increase of potassium in males receiving 800mg/kg/day (40% and 9%, respectively). Due to the low severity, these findings were considered to be not adverse.
Recovery phase
Cholesterol was comparable with controls, confirming reversibility. The statistically significant differences recorded (triglycerides and sodium in females) were not observed at the end of dosing, therefore considered unrelated to treatment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Dosing phase
Augmented diuresis was recorded in males dosed at 240 and 800mg/kg/day and in the
female receiving 800mg/kg/day. Compared with controls, the increments were 2.1 and
3.2 fold, respectively, in males and 2.1 fold in the female. In the absence of other related
findings, this change was considered of no toxicological significance.
Recovery phase
Diuresis in males showed a trend to reversibility
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
moderate and statistically significant increase in landing footsplay and a slight statistically
significant decrease in grip strenght were recorded in female animals of Groups 2 and 3
at the end of treatment period. In the same occasion, a slight and statistically significant
reduction in grip strenght was observed in females of Groups 2 and 3. Although data were
not available for females of Group 4, due to their premature death, these changes could
be considered treatment-related, but not adverse. In fact, these changes were slight and
amply within historical control data and theywere no longer apparent at the end of recovery
period.
A moderate and statistically significant increase in motor activity was recorded in treated
male animals at the end of dosing period, when compared to controls, although this change
was not dose-related. No differences between animals of the treatment groups (Groups 2
and 3) and control animals were recorded at the end of recovery period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Final sacrifice
Terminal body weight did not show any relevant changes in treated males and females,
when compared to their controls.
Treatment-related organ weight changes were noted in males and females dosed at ≥ 240mg/kg/day. In particular, males dosed at 240mg/kg/day showed an increase in the
absolute and relative kidneys weights. The liver weight was increased in males, statistically
significant in males dosed at ≥ 80mg/kg/day for the absolute weight, while only in males
dosed at 800mg/kg/day for the relative (to body weight) weight. A minimal increase in
kidneys and liver weights was also noted in females dosed at ≥ 240mg/kg/day. A minimal
reduced absolute and relative thymus weight was observed in treated males and females.
Recovery sacrifice
Terminal body weight did not show any relevant changes in treated males and females
when compared to their controls. Relative kidneys weight was still statistically significantly
increased in males dosed at 80mg/kg/day. No other relevant changes were noted in treated
animals, when compared with controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Final sacrifice
At post mortem examination, no treatment-related changes were observed. A subcutaneous
mass in a low dose female or a reduced size of the thymus in few high dose males or some
control and treated females were the only relevant changes noted. Reduced thymus size were also present in control and satelite controls with high numbers, the finding could be considered stress-related rather than treatment-related.
Recovery sacrifice
At post mortem examination, no treatment-related changes were observed.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Final sacrifice
Treatment-related histopathological findings were seen in the liver, kidneys and thymus of
male and female animals in all treatment groups, as described below:
– Liver:centrilobular hepatocytic hypertrophy was noted in males and females dosed
at 240 and 800mg/kg/day (Groups 3 and 4), but not in male and female animals
receiving the test item at 80mg/kg/day (Group 2). This change was represented
by an enlargement of individual hepatocytes, with fine granular eosinophilic cytoplasm.
Such liver pathology, correlated with the increased organ weight and with a
dose related trend, was considered a functional adaptative change that enhanced, by
enzymes and trasporters, the metabolism and excretion of the xenobiotic. No degenerative
changes such as necrosis of hepatocytes (including monocellular necrosis) or
relevant inflammatory responses were seen.
- Kidneys: increased severity and/or incidence of nephropathy in males and females
dosed at ≥ 240 mg/kg/day, when compared with their controls, mainly represented
by degenerative tubules, in some instances involving the entire nephron, in both
the cortex and medulla. Renal tubules showed dilatation filled with proteinaceous
casts, especially at the corticomedullary junction and/or were lined by epithelium
with basophilic cytoplasm, whereas other tubules were lined by atrophic, flattened
epithelium in some instances associated by interstitial cellular infiltration.
Males and females receiving Bis(2-(2-butoxyethoxy)ethoxy) methane at 80 mg/kg/day
(Group 2) showed a similar incidence and/or severity to their controls. Considering that
nephropathy is one of the mostcommonspontaneous lesions and the single most important
renal disease in rats, in particular Sprague Dawley strain the minimal variation between
control and low dose (80 mg/kg/day) animals it could be considered incidental rather than
clearly treatment-related.
– Thymus: atrophy, mainly noted in high dose males and in most females dosed at
80mg/kg/day with an increased incidence and/or severity when compared to controls
and was characterised by a reduced thymic cellularity in particular in the cortex,
however considering that some control males and females showed a similar pathological
picture, the finding could be considered stress-related rather than treatment-related.
Due to the high variation within the dosegroups and considering that controls and sattelite
controls showed similar pattern than 80 mg/kg bw/d group the effect is judged stress related
and not adverse at this dose level.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle
and to the integrity of the various cell types within the different stages; regular layering in
the germinal epithelium was noted.
Normal physiology of the estrous cycle (estrus, metestrus, diestrus and proestrus) was
noted in control and treated females. The morphological changes seen were normal when
compared to each “estrous phase” in the ovaries, uterus/cervix and vagina. The remaining
sporadic lesions, reported in control and treated animals, were considered to be an expression
of spontaneous and/or incidental pathology, seen in this species and age of untreated
animals.

Recovery sacrifice
Treatment-related changes were only noted in the kidneys of males and females dosed at
240 mg/kg/day.
Renal nephropathy were still recorded with and increased incidence and severity mainly in
males and females receiving the test item at 240 mg/kg/day.
Atrophy of thymus was noted in most females and in few males dosed at 240 mg/kg/day,
however considering that some control males and females showed a similar pathological
picture, the finding could be considered stress-related rather than treatment-related.
The liver show a complete reversibility in Group 3 males and females.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormone determination
Dosing Phase
Increments of 2.0 to 2.8 fold when compared to control animals, with some dose-relation, were recorded in thriiodothyronine (T3) in three male animals of Group 4 , treated at 800mg/kg/day, and in one male of Group 3 dosed at 240mg/kg/day. This animal showed a decrement in thyroid stimulating
hormone (TSH) of 71% when compared to control values. TSH was also decreased in one animal of Group 4 (-66% in comparison to controls). The findings above described were considered to be incidental, since similar values were recorded also in one control animal, due to the inconsistency between sexes
and the absence of histopathological changes involving the thyroid.

Recovery phase
No changes were recorded.
Details on results:
Oestrous cycle
Evaluation of the oestrous cycle at the end of treatment period did not indicate any significant
differences between groups
Key result
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
240 mg/kg bw/day (actual dose received)
System:
other: kidney
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
The dose level of 80 mg/kg/day could be considered the NOAEL (No Adverse Effect Level) in this study.
Executive summary:

The toxicity of Bis(2-(2-butoxyethoxy)ethoxy)methane in rats after oral administration for 13 weeks and recovery from any treatment-related effects during a period of 4 weeks, were investigated in this study, according to OECD 408 guideline.

Three groups, each of 10 male and 10 female Sprague Dawley rats, received the test item by gavage at dosages of 80, 240 and 800mg/kg body weight/day for 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (aqueous solution of carboxymethylcellulose 0.5% w/v) and acted as a control. Five additional animals for each sex were

included in each group for recovery assessment.

The following investigations were performed: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli and motor activity, body weight, food consumption, ophthalmoscopy, clinical

pathology investigations (including thyroid hormone determination), terminal bodyweight, organ weights, macroscopic observations, histopathological examination.

Severe clinical signs (including an high incidence of mortality) were observed at the high dose level of 800 mg/kg/day and, with lower severity and incidence, at the mid-dose of 240 mg/kg/day.

No significant clinical signs or other signs of toxcity were observed in animals dosed at 80 mg/kg/day during the in vivo phase of the study.

At post mortem, changes in the liver at 240 and 800 mg/kg/day were considered to adaptive.

Atrophy of the thymus was mainly reported in high dose males and in most females dosed at 80 mg/kg/day at the end of treatment period, with an increased incidence and/or severity,

where compared to controls. At the end of recovery period, atrophy of the thymus was noted in controls and some treated animals. Therefore, this change could be considered stress-related rather than

treatment-related and, considered the high variability noted at the end of treatment period, it is considered not adverse/significant at 80 mg/kg/day.

The nephropathy observed in animals dosed at ≥ 240 mg/kg/day at the end of treatment period showed an increase in incidence and severity in male and female animals treated at

240 mg/kg/day at the end of recovery, when compared to controls, while at the dose level of 80 mg/kg/day, the incidence and severity were comparable between controls and treated

animals.

Therefore, the dose level of 80 mg/kg/day could be considered the NOAEL (No Adverse Effect Level) in this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Organ:
kidney
liver

Additional information

Oral:

OECD 422:

A GLP conform repeated dose/ reproductive toxicity study was performed with TP-90B RUBBER CHEMICAL according to OECD 422 (RTC/ Rohm & Haas 2004). Groups of 10 Sprague-Dawley rats per sex received TP-90B Rubber Chemical in 0.5% aqueous carboxymethylcellulose at doses of 10, 100 and 800 mg/kg bw/d by gavage. Males were treated for 2 weeks prior to pairing, through pairing until the day before sacrifice. Females were treated for 2 weeks prior to pairing, through the mating and gestation periods up to day 3 of lactation. Dams and offspring were sacrificed on Day 4 post-partum (= at least 43 days). Observations were made as follows:

Fate of females

One mid dose female was sacrificed on Day 0 post-partum for human reasons since signs of dystocia (difficult delivery) were noted.

Weekly physical examinations including detailed clinical signs and neurotoxicity assessment (Functional Observation Battery) did not show any treatment-related effects. Daily post-dose observations of clinical signs showed high dose male animals with ataxia,

Semi closed eyes, hunched posture, reduced activity and salivation. Pronation, ataxia, semiclosed eyes, reduced activity, hunched posture, lethargy, twitches, piloerection and salivation were noted at post-dose observations in high dose females.

Body weight and body weight gain

Slight decreases in body weight and body weight gain occurred only in high dose females during gestation.

Food consumption

Food consumption remained comparable between control and treated groups in both sexes before pairing. Reduced food consumption was noted in high dose females on Days 7 and 14 post-coitum.

Motor activity and sensory reaction to stimuli

Motor activity measurements and sensory reaction to stimuli were unaffected by treatment at any dose level in both sexes.

Haematology

No toxicologically significant change in haematology values occurred in males or females at any dose level.

Clinical chemistry

No toxicologically significant change in any clinical chemistry parameter occurred in malesor females at any dose level.

Terminal body weight and organ weights

No differences in terminal body weights were observed between treated and control animals. Absolute and relative liver weights were increased in high dose males and females when compared to controls. All changes were statistically significantly different from controls except the absolute liver weight in females.

Macroscopic examination

No macroscopic change was reported at necropsy in the examined organs/tissues of the animals killed at termination that could be considered related to the administration of the test item. The gross changes observed were considered to be incidental or spontaneous in origin.

Microscopic examination

No microscopic changes clearly related to test item administration were observed in male or female rats in the low and mid-dose groups. Diffuse hepatocellular hypertrophy (enlarged hepatocytes in all areas of the lobules) occurred in male and female rats of the high dose group. A finding of uncertain significance was observed in the high dose group. Focal germ cell depletion and degeneration in one testis (unilateral) occurred in four high dose male rats. Three of these four rats also had a few exfoliated spermatogenic cells in the lumen of the epididymal tubules.

In conclusion, following effects were observed only at the high dose (800 mg/kg bw/ day):

- Post-dose clinical signs indicating general effects of toxicity in males and females

- Decreased body weight and body weight gain in females during gestation

- Reduced food consumption in females on days 7 and 14 post-coitum

- Increased liver weights

- Microscopic changes in the liver

The NOAEL of this study was 100 mg/kg bw/d since increases in absolute and relative kidney weights noted in the mid-dose male group only were not interpreted as a clear adverse health effect as this effect was not observed in the high-dose group.

OECD 408:

The toxicity of Bis(2-(2-butoxyethoxy)ethoxy)methane in rats after oral administration for 13 weeks and recovery from any treatment-related effects during a period of 4 weeks, were investigated in this study, according to OECD 408 guideline.

Three groups, each of 10 male and 10 female Sprague Dawley rats, received the test item by gavage at dosages of 80, 240 and 800mg/kg body weight/day for 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (aqueous solution of carboxymethylcellulose 0.5% w/v) and acted as a control. Five additional animals for each sex were

included in each group for recovery assessment.

The following investigations were performed: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli and motor activity, body weight, food consumption, ophthalmoscopy, clinical

pathology investigations (including thyroid hormone determination), terminal bodyweight, organ weights, macroscopic observations, histopathological examination.

Severe clinical signs (including an high incidence of mortality) were observed at the high dose level of 800 mg/kg/day and, with lower severity and incidence, at the mid-dose of 240 mg/kg/day.

No significant clinical signs or other signs of toxcity were observed in animals dosed at 80 mg/kg/day during the in vivo phase of the study.

At post mortem, changes in the liver at 240 and 800 mg/kg/day were considered to adaptive.

Atrophy of the thymus was mainly reported in high dose males and in most females dosed at 80 mg/kg/day at the end of treatment period, with an increased incidence and/or severity,

where compared to controls. At the end of recovery period, atrophy of the thymus was noted in controls and some treated animals. Therefore, this change could be considered stress-related rather than

treatment-related and, considered the high variability noted at the end of treatment period, it is considered not adverse/significant at 80 mg/kg/day.

The nephropathy observed in animals dosed at ≥ 240 mg/kg/day at the end of treatment period showed an increase in incidence and severity in male and female animals treated at

240 mg/kg/day at the end of recovery, when compared to controls, while at the dose level of 80 mg/kg/day, the incidence and severity were comparable between controls and treated

animals.

Therefore, the dose level of 80 mg/kg/day could be considered the NOAEL (No Adverse Effect Level) in this study.

 

dermal:

no data available

 

 

inhalation

no data available

 


Repeated dose toxicity: via oral route - systemic effects (target organ) liver and kidney

Justification for classification or non-classification

Based on the available data on the repeated dose toxicity of hexaoxatricosane no classification is warranted according to the criteria of both, 67/548/EEC and CLP regulation (EC) 1272/2008.