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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 Feb - 21 Oct 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom, Medicines & Healthcare Products Regulatory Agency, UK
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Propylidynetrimethanol, propoxylated
EC Number:
500-041-9
EC Name:
Propylidynetrimethanol, propoxylated
Cas Number:
25723-16-4
Molecular formula:
C3H5(CH2OR)3 R= (C2H3(CH3)O)nH sum of n: >1 - <6.5 mol PO
IUPAC Name:
Propylidynetrimethanol, propoxylated
Test material form:
liquid
Specific details on test material used for the study:
Materail name: TMP-PO

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The species and strain chosen are the accepted rodent species and strain for nonclinical toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent, UK
- Age at study initiation: 8 weeks
- Weight at study initiation: 216 - 296 g (males) and 138 - 201 g (females)
- Housing: Up to 3 animals of each sex per dosing group in polycarbonate cages with stainless steel grid tops and solid bottoms; appropriate bedding was provided.
- Diet: Special Diet Services Rat and Mouse No. 1 Diet SQC, ad libitum; supplier: Special Diet Services, Witham, England
- Water: Public tap water, ad libitum
- Acclimation period: up to 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 41 - 69
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: To: 24 Mar - 23 Jun 2020

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral (gavage) route of exposure was selected because it is accepted to be the most appropriate route of administration for this kind of study.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared weekly by dissolving appropriate amounts of the test material in water yielding a final concentrations of 10, 30 and 100 mg/mL. The solutions were stored in a refrigerator set to maintain 4°C, protected from light, and dispensed daily.

VEHICLE
- Concentration of test substance in vehicle: 10, 30 and 100 mg/mL
- Dose volume: 10 mL/kg bw
- Lot/batch no.: Not applicable, continously prepared by a Mili-Q system
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations were analysed for concentration, homogeneity and stability by gas chromatography (GC) with flame ionisation detection (FID) using a validated analytical procedure (Jaradi, 2020, study no. 424083). Concentration and homogeneity determinations yielded acceptable results (for concentration: mean sample concentration results within or equal to ± 15% of theoretical concentration, each individual sample concentration result within or equal to ± 20%; for homogeneity: relative standard deviation (RSD) of concentrations of <= 10% for each group). The stability analyses performed demonstrated that the test substance is stable in the vehicle.
Duration of treatment / exposure:
at least 90 days
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a foregoing range finding study, in which animals were orally exposed to 100, 300 and 1000 mg/kg bw/day for 28 days. Clinical signs of toxicity were noted at 300 or 1000 mg/kg bw/day but no further signs of toxicity were noted at any dose level. Therefore, 100, 300 and 1000 mg/kg bw/day were selected as the dose levels for the main study.
- Fasting period before blood sampling for clinical biochemistry: Overnight with a maximum of 24 h

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS, MORTALITY AND POSTDOSE OBSERVATIONS: Yes
- Time schedule: At least once daily before animals were dosed, beginning in the week before dosing commenced.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly, from the week before dosing commenced and throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly, from the week before dosing commenced and throughout the study.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, food consumption determined at least weekly, from the week before dosing commenced and throughout the study, quantitatively measured per cage.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Checked on regular basis throughout the study, monitored by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule and dose groups for examinations: Pretreatment: once (all animals), dosing period: week 13 (all animals of control and high-dose groups).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination of study.
- Anaesthetic used for blood collection: Yes (isofluorane).
- Animals fasted: Yes (overnight, max. 24 h).
- How many animals: All
- Parameters examined: Red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, red blood cell distribution width, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, reticulocyte count (absolute), platelet count, white blood cell count, neutrophil count (absolute), lymphocyte count (absolute), monocyte count (absolute), eosinophil count (absolute), basophil count (absolute), large unstained cells (absolute), other cells (as appropriate).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination of study.
- Animals fasted: Yes (overnight, max. 24 h).
- How many animals: All
- Parameters examined: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, creatine kinase, total bilirubin, urea, creatinine, calcium, phosphate, blood urea nitrogen (BUN), total protein, albumin, globulin (calculated), albumin/globulin ratio, glucose, total Cholesterol, HDL and LDL cholesterol, triglycerides, sodium, potassium, chloride.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule and dose groups for examinations: Once during the dosing period, 5 animals per sex per group in Week 13.
- Battery of functions tested: sensory activity / grip strength / motor activity / pain perception / landing foot splay.

IMMUNOLOGY: No

OTHER:
- Detailed Functional Observations: Once during pretreatment and once weekly during the dosing period for all animals, incl. posture/condition on first approach (animal undisturbed), checking prostration, sterotypy / bizarre behaviour, tremors (head, limbs, whole body), convulsions, ease of removal from the cage, body temperature, rectal temperature, condition of the eyes, body tone. pinna response, presence of salivation, overall ease of handling, respiration rate and pattern, air righting, extensor thrust, observations in a standardised arena (2 min observation period).
- Monitoring of Estrous Cycle: Vaginal smears of all females were examined on the morning of necropsy.
- Thyroid hormones: TSH, T3 and T4 (on morning of necropsy after overnight fasting on all animals).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organs and tissues: Artery (aorta), body cavity (nasal), bone marrow (sternum), bone marrow smear, bone (sternum), brain, epididymides, esophagus, eye, ganglion (dorsal root, lumbar), glands (adrenal, clitoral, lacrimal, Harderian, mammary, parathyroid, pituitary, preputial, prostate, salivary (submandibular, sublingual and parotid), seminal vesicle, thyroid, Zymbal’s), gut-associated lymphoid tissue, heart, kidney, large intestine (cecum, colon and rectum), larynx, liver, lung, lymph node (mandibular and mesenteric), skeletal muscle, nerve (optic, sciatic and tibial), ovary, oviduct, pancreas, skin, small intestine (duodenum, ilium and jejunum), spinal cord, spleen, stomach, testis, thymus, tongue, trachea, ureter, urinary bladder, uterus/cervix, vagina

HISTOPATHOLOGY: Yes
- Like above EXCEPT: Bone marrow smear, ganglion (dorsal root, lumbar), glands (clitoral, lacrimal, Harderian, preputial, salivary, Zymbal's), nerve (tibial), oviduct, ureter
Other examinations:
Monitoring of Estrous Cycle: Vaginal smears on all females were examined on the morning of necropsy to determine the stage of estrous cycle to allow correlation with histopathology of ovaries.

Sperm Evaluation:
- Computer-aided sperm assessment (CASA): All males, an appropriate sperm suspension was examined using a Hamilton Thorne sperm motility analyser
- Sperm count and morphological analysis: Sperm suspension were counted using a haemocytometer, a total sperm count expressed per cauda epididymis and per gram of cauda epididymis was obtained; a sperm smear was prepared and stained with eosin Y solution, from the control and high-dose group males at least 200 sperm per animal were evaluated for morphological abnormalities
- Spermatid count: A suspension was prepared by homogenising the right testes, the number of homogenisation-resistant spermatids were counted using a haemocytometer to obtain a total spermatid count expressed per testis and per gram of testis
Statistics:
Constructed Variables:
Body Weight Gains: Calculated between each scheduled interval
Organ Weight Relative to Body Weight: Calculated against the terminal body weight
Organ Weight Relative to Brain Weight: Calculated against the brain weight

Descriptive Statistical Analyses:
Means, standard deviations, percentages, numbers, and/or incidences have been reported as appropriate by dataset.

Inferential Statistical Methods:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and have been reported at the 1% and 5% levels.

Parametric/Non-parametric
Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. Datasets with two groups were compared using a Dunnett’s test (equivalent to t-test in Nevis 2012 tables) or Dunn’s test (equivalent to Wilcoxon Rank-Sum test in Nevis 2012 tables).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Irregular respiration, uncoordinated behavior, ploughing and animals eating bedding during the first few weeks of dosing (adverse); salivation in all animals up to 1 h post dose throughout the study (probably due to the taste of the test substance); sporadic hunched posture, partly closed eyes, irregular/abnormal respiratory rate, locomotor, subdued behavior, lying on side and erect fur in individual male and female animals on single days (adverse).
300 mg/kg bw/day: One male was eating bedding and two females were noted with salivation (probably due to the taste of the test substance).
Mortality:
mortality observed, treatment-related
Description (incidence):
1000 mg/kg bw/day: One female was prematurely euthanised on Day 11 due to adverse clinical signs (abnormal gait, irregular and labored breathing, partly closed eyes, decreased activity, uncoordinated behavior, hunched posture, erect fur and tremors).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
≥100 mg/kg bw/day: Reduced body weight and body weight gain in males (non-adverse).
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
≥100 mg/kg bw/day: Increased white blood cell counts (lymphocytes, basophils and large unstained cells) in males and females (non-adverse).
≥300 mg/kg bw/day: Increased fibrinogen in males and females (non-adverse).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
≥100 mg/kg bw/day: Increased triglycerides, cholesterol, total protein and globulin resulting in a lower A/G ratio in males and females (non-adverse).
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Reduction in basic and fine motor movements during functional observation in females (non-adverse).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
300 and 1000 mg/kg bw/day: Increased liver weights (non-adverse); increased kidney weights in males (equivocal).
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Hepatocyte hypertrophy (non-adverse).
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No effects were observed on oestrus cycle and sperm analysis.
Details on results:
Mortality:
One female dosed at 1000 mg/kg bw/day was prematurely euthanised on Day 11 due to adverse clinical signs including abnormal gait, irregular and labored breathing, partly closed eyes, decreased activity, uncoordinated behavior, hunched posture, erect fur and tremors.

Clinical signs:
At 1000 mg/kg bw/day, salivation was noted in all animals up to 1 h post dose throughout the study. In addition, clinical signs including irregular respiration, uncoordinated behavior, ploughing and animals eating bedding were noted during the first few weeks of dosing. Sporadic hunched posture, partly closed eyes, irregular/abnormal respiratory rate, locomotor, subdued behavior, lying on side and erect fur on Day 66 were noted in one female and hunched posture was noted in one male on Day 65 only. At 300 mg/kg bw/day, one male was eating bedding and two females were noted with salivation. These signs are considered to be related to the possible taste of the test substance

Functional observations:
Uncoordinated behaviour and a reduction in basic and fine motor movements were noted during the functional observations in females at 1000 mg/kg bw/day. This finding was found to be transient in nature, lacked statistical significance and therefore considered non-adverse.

Body weight development:
Dose related decreased body weight and a decreased body weight gain was noted in males at the end of the study at ≥100 mg/kg bw/day, when compared with controls. However, there were no correlating changes in food consumption and animals still gained weight therefore this was considered to be non-adverse.

Haematology and clinical chemistry:
Increased white blood cell counts mainly attributable to lymphocytes, basophils and large unstained cells were noted in males and females at ≥100 mg/kg bw/day. Increased fibrinogen was noted in males and females receiving ≥300 mg/kg bw/day and clinical chemistry changes included increased triglycerides, cholesterol, total protein and globulin resulting in a lower A/G ratio were noted in males and females receiving ≥100 mg/kg bw/day. These findings lacked dose dependency, were small in magnitude, and/or had no microscopic correlates. Therefore, they were considered to be non-adverse.

Organ weights and histopathology:
There were treatment-related increased liver weights at 300 and 1000 mg/kg bw/day associated with microscopic findings in the liver (hepatocyte hypertrophy) at 1000 mg/kg bw/day only. These findings were considered to be adaptive changes and thus, non-adverse. There were also increased kidney weights at 300 and 1000 mg/kg bw/day (in males only), in the absence of any microscopic findings and correlating clinical pathology changes, the toxicological significance of this findings is considered equivocal.

Food consumption:
No changes were noted.

Ophthalmology:
There were no changes observed.

Thyroid hormone analysis:
Treatment with the test substance did not change thyroid hormone levels.

Oestrus cycle:
The test substance did not exert an effet on the oestrus cycle.

Sperm analysis:
No effects were noted in the sperm analysis.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
other: One unscheduled death and clinical signs in surviving animals including uncoordinated behaviour and irregular respiration reported at the highest dose level of 1000 mg/kg bw/day.
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
other: One unscheduled death and clinical signs in surviving animals including uncoordinated behaviour and irregular respiration reported at the highest dose level of 1000 mg/kg bw/day.

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: based on clinical signs and mortality, no target system or organ identified
Organ:
other: based on clinical signs and mortality, no target system or organ identified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Due to the amount of supporting information, all data necessary to back-up the information reported in this endpoint study record are attached as pdf document under 'Attached background material'.

Applicant's summary and conclusion