Reaction mass of 1,3-dioxane-5,5-dimethanol and 1,3-Propanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 August 2015 to 30 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Modern GLP and guideline-compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF No. 12-Nousan-8147

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Polyol PX

IUPAC Name:

Polyol PX

Constituent 2

Reference substance name:

Reaction mass of 1, 3-Propanediol, 2-(Hydroxymethyl)-2- [(Methoxymethoxy) methyl]- and 1,3- Dioxane- 5,5- Dimethanol.

IUPAC Name:

Reaction mass of 1, 3-Propanediol, 2-(Hydroxymethyl)-2- [(Methoxymethoxy) methyl]- and 1,3- Dioxane- 5,5- Dimethanol.

Constituent 3

Reference substance name:

Reaction mass of 1,3-Propanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-dioxane-5,5-dimethanol

EC Number:

911-819-6

Cas Number:

68442-60-4

IUPAC Name:

Reaction mass of 1,3-Propanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-dioxane-5,5-dimethanol

Test material form:

other: solid or melt product

Details on test material:

- Name of test material (as cited in study report): Polyol PX - Reaction mass of 1, 3-Propanediol, 2-(Hydroxymethyl)-2-
[(Methoxymethoxy) methyl]- and 1,3- Dioxane- 5,5-Dimethanol.
- Physical state: solid or melt product
- Analytical purity: 83.9% (corrected for purity; correction factor 1.19)
- Lot/batch No.: 441258479C
- Expiration date of the lot/batch: 02 June 2016
- Stability under test conditions: confirmed by the sponsor
- Storage condition of test material: Ambient in the dark

Specific details on test material used for the study:

- Name of test material (as cited in study report): Polyol PX; alternative name: Reaction mass of 1, 3-Propanediol, 2-(Hydroxymethyl)-2-[(Methoxymethoxy)methyl]-and 1, 3-Dioxcine 5, 5-Dimethanol
- Source: Sponsor
- Physical state: Solid
- Purity: 83.9% (dose calculations were corrected for purity; correction factor 1.19)
- Lot/batch No.: 441258479C
- Expiration date of the lot/batch: 02 June 2016

Stability analyses performed previously in conjunction with Test Facility Study No. 425834
(Cockburn, P.2010) demonstrated that the test item was stable in the vehicle when prepared
and stored under the same conditions at concentration range of 2-200 mg/mL. Stability data
have been retained in the study records for Test Facility Study No. 425834

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: The animals were Han Wistar rats
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: 8-9 weeks old
- Weight at study initiation: 227-285 g (males); 166-167 g (females)
- Housing: 2/3 per cage by sex in polycarbonate cages with stainless steel grid tops and solid bottoms
- Diet (e.g. ad libitum): Rat and Mouse (modified) No. 1 Diet SQC Expanded (Special Diet Services, Witham, Essex), ad libitum
- Water (e.g. ad libitum): water from the public supply ad libitum (Scottish Water, Edinburgh, Midlothian, UK)
- Acclimation period: up to 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25 °C
- Humidity (%): 40-64%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 13 August 2015 To: 20 November 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dose level requirements. The appropriate amount of test item was weighed and the appropriate volume of vehicle was added to the final volume. The solution was mixed by magnetic stirring until a visibly homogenous yellowish solution was obtained (sonication was used incorrectly on two occasions but an acceptable formulation was obtained and this was not considered to have affected the study). The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and stirred for at least 30 min before dosing. The dosing formulation was stirred continuously during dosing. Any residual volumes were discarded.

The dosing volume was 10 mL/kg (five control animals received 15 mL/kg in error on one occasion, but as this was the control substance (water) the deviation was not considered to affect the validity of the study)

VEHICLE
- Concentration in vehicle: 0, 11.9, 35.7, 119 mg/mL (corrected for purity)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration and homogeneity analyses were performed from all dosing formulations during Week 1, Week 6 and Week 12 by HPLC (reverse phase) with ELSD detection using a validated analytical procedure.

Duplicate top, middle and bottom samples (duplicate middle only for control) of 1 mL for each sampling time point were collected and sent to the analytical laboratory for analysis. Triplicate sets of top, middle and bottom samples (middle only for control) of the same volume were also collected and retained as backup samples. Concentration results were considered acceptable if mean sample concentration results were within 10% of theoretical concentration. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤5% for each group.

Stability analyses performed previously in conjunction with Test Facility Study No. 425834 (Cockburn, P.2010) demonstrated that the test item was stable in the vehicle when prepared and stored under the same conditions at concentration range of 2-200 mg/mL.

Duration of treatment / exposure:

91 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected following evaluation of a previous 14 day repeated dose oral (gavage) toxicity study in the rat (Charles River Study No. 528596) performed on behalf of the Sponsor. The high dose level was expected to produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal to moderate toxic effects. The low dose level was expected to produce no observable indications of toxicity.

Positive control:

Not required.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily; early morning and as late as possible for each day. Animals were also examined for reaction to treatment regularly throughout the day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretreatment and daily throughout dosing.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Regular visual inspection of water bottles

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretreatment for all animals, and during Week 13 for control and high dose animals. An indirect ophthalmoscope was used for examination after the application of the mydriatic agent (1% Tropicamide, Mydriacyl)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 14 (prior to euthanasia)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all (10/sex/group)
- Parameters examined: Approximately 0.5 mL of blood was collected into tubes containing K2EDTA and were analysed for: Red blood cell count, Haemoglobin, Haematocrit, Mean cell volume, Mean cell haemoglobin concentration, Mean cell haemoglobin, Reticulocytes, Reticulocyte count (absolute), Red blood cell distribution width, Platelets, White blood cell count, Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Large unstained cells. A blood smear was prepared from each haematology sample. Blood smears were labelled, stained, and stored. Approximately 0.9 mL of blood was collected into tubes containing 0.1 mL 3.8% (w/v) trisodium citrate. Blood samples were processed to plasma, and analysed for Activated partial thromboplastin time, Fibrinogen, Prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 14 (prior to euthanasia)
- Animals fasted: No
- How many animals: all (10/sex/group)
- Parameters examined: Approximately 1.0 mL of blood was collected and into tubes containing lithium heparin. Blood samples were processed to plasma, and analysed for: Urea, Glucose, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Creatine phosphokinase, Lactate dehydrogenase, Sodium, Potassium, Chloride, Total protein, Albumin, Globulin, Albumin/globulin ratio, Cholesterol, Creatinine, Total bilirubin, Calcium, Inorganic phosphate, Triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters examined: Microscopic evaluation of spun deposit, Colour, Turbidity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Leukocytes, Blood Pigments, Urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during pretreatment and once during Weeks 1, 4, 8 and 12/13
- Dose groups that were examined: All
- Battery of functions tested: see below

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The following organ weights were obtained: brain, epididymis, adrenal gland, pituitary gland, prostate, thyroid, heart, kidney, liver, lung, ovary, spleen, testis, thymus, uterus. Paired organs were reported together. Organs were weighed before fixation. Organ to body weight ratios/percentages (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes; representative samples from the following list were collected from all animals and preserved in 10% neutral buffered formalin, except for the eye and optic nerve which were preserved in Davidson's fixative and the testis which was preserved in Modified Davidson's fixative: animal identification, aorta, bone marrow smear, bone marrow (femur and sternum), bone (femur and sternum), brain, cervix, epididymis x2, eye x2, adrenal gland x2, harderian gland x2, lacrimal gland x2, mammary gland, parathyroid gland x2, pituitary, prostate, salivary gland x2, seminal vesicle, thyroid x2, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney x2, caecum, colon, rectum, liver, lung, mandibular lymph node, mesenteric lymph node, skeletal muscle, nasal cavity, optic nerve x2, sciatic nerve x2, oesophagus, ovary x2, oviduct x2, pancreas, pharynx, skin, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testis x2, thymus, tongue, trachea, ureter x2, urinary bladder, uterus, vagina. Samples were embedded in paraffin, sectioned, mounted on glass slides and stained with haematoxylin and eosin. Duplicate bone marrow smears were taken and stained using May-Grunwald-Giemsa. No evaluation of the smears was made, however slides were retained.

Other examinations:

No other examinations reported.

Statistics:

Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females were analysed separately. Pairwise comparisons were only performed against the control group (Group 1). Body weight, food consumption, selected functional observational battery and motor activity data, haematology, coagulation, clinical chemistry and selected urinalysis data were analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test i.e. pairwise comparisons were made only if the overall F-test is significant. If the variances were heterogeneous, log or square
root transformations were used in an attempt to stabilise the variances. If the variances remain heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test). In circumstances where it was not possible to perform the F Max test due to zero standard deviation in at least one group, the non-parametric ANOVA results were reported.
Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA as above as an exploratory analysis. In circumstances where the variances in the ANCOVA remain heterogeneous following log or square root transformations, the data was subjected to a rank transformation prior to analysis.
In the ANOVA and ANCOVA summary tables, the results of the analysis were reported indicating the level of statistical significance (p<0.05, p<0.01 and p<0.001) of each pairwise comparison. Actual p-values were not reported in the summary tables for these analyses.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

one animal in the 1000 mg/kg bw/d group was euthanised on Day 54, however the death was not considered related to treatment

Mortality:

mortality observed, non-treatment-related

Description (incidence):

one animal in the 1000 mg/kg bw/d group was euthanised on Day 54, however the death was not considered related to treatment

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

except for the animal that was euthanised: not considered to be related to treatment

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

not considered to be related to treatment

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

not considered to be related to treatment

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

not considered to be related to treatment

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

except for the animal that was euthanised: not considered to be related to treatment

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY
One male in the 1000 mg/kg bw/d group (animal 404M) was euthanized on Day 54 of the study due to poor overall condition including weight loss (displayed a reduction in bodyweight from Day 52 until unscheduled euthanasia on Day 54), behavioural changes (ploughing its muzzle through the substrate, subdued and reduced activity) and irregular respiration. On microscopic evaluation, arteritis/periarteritis in the brain, heart and kidney, myocardial degeneration, haemorrhage in the brain, nephropathy and nephroblastoma were noted. These findings most likely caused the weight loss observed in this animal, however, due to the lack of treatment related lesions, these observations were considered spontaneous and unrelated to the administration of Polyol PX.

CLINICAL SIGNS
There were no adverse treatment-related clinical observations evident in either males or females that received up to 1000 mg/kg bw/d of Polyol PX.
There were no adverse clinical observations noted during treatment that were considered due to treatment with Polyol PX. Transient incidents of minor behavioural changes were observed where an animal ploughed its nose through its substrate and bedding immediately post dose was noted in 1/10 males and 3/10 females that received 1000 mg/kg/day; incidences were transient and observed on 1 or 2 occasions throughout treatment. Additionally, increased vocalisation was observed in Animal 257F that received 300 mg/kg bw/d from Day 28 and until study completion. As this was an isolated incident and lacked any dose-relationship, coupled with the absence of any treatment related findings, this was considered to be due to individual animal variation. All other clinical observations were considered typical of animals of this strain and age on this type of study conducted at the Test Facility.

BODY WEIGHT AND WEIGHT GAIN
Body weight profiles were unaffected by administration of Polyol PX in animals that received up to and including 1000 mg/kg bw/d.

FOOD CONSUMPTION
Food consumption was unaffected in males receiving Polyol PX up to and including 1000 mg/kg bw/d,whereas during the first 5 to 6 weeks of treatment, the quantity of food consumed for females receiving 1000 mg/kg bw/d was slightly lower when compared to concurrent controls, achieving statistical significance on occasion. Although slightly lower than controls, as body weight profiles were unaffected this minor difference was considered
toxicologically insignificant.

WATER CONSUMPTION
Visual inspection of the water bottles indicated no observable differences in water consumption between groups throughout the treatment period.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related findings.

HAEMATOLOGY
There were no haematology or coagulation parameter findings that were considered to be related to the administration of Polyol PX during the treatment period.

CLINICAL CHEMISTRY
There were minor changes in clinical chemistry parameters (slightly high cholesterol and total protein in males that received 1000 mg/kg bw/d) noted during the treatment period however, any differences in the parameters were within the range of the overall control group data, or were judged to be due to individual changes. Additionally, as the differences lacked a true dose-relationship they were therefore considered un-related to treatment with Polyol PX.

URINALYSIS
There were minor changes in urinalysis parameters (slightly high urinary volume and specific gravity in males and females that received 1000 mg/kg bw/d and high urinary volume in females that received 300 mg/kg bw/d noted during the treatment period however, any differences in the parameters were generally within the range of the overall control group data, or were judged to be due to individual changes. Additionally, as the differences lacked a true dose-relationship they were therefore considered un-related to treatment with Polyol PX.

NEUROBEHAVIOUR
There were no treatment-related differences in the functional observation battery parameters, quantitative functional observation parameters or motor activity following administration of Polyol PX at up to 1000 mg/kg bw/d. A difference in the general behaviour of the animal in their home cage was observed throughout the assessment occasions, and in all treated animals up to and including 1000 mg/kg/day where the number of animals that were asleep/at rest was increased and/or decreased when comparison to controls. Although consistently different throughout treatment, these were also different when compare to pre-treatment observations. As there were no abnormal observations noted on the behaviour of animals throughout treatment these differences were considered to be incidental and due to inter-animal variation. All of the behaviours observed were considered typical for rats of this age and strain on this type of study conducted at the Test Facility.

ORGAN WEIGHTS
No treatment-related organ weight changes were noted. There were isolated organ weight values that were different from the controls, however as there were no trends/patterns or correlating data they were considered un-related to treatment.

GROSS PATHOLOGY
No treatment-related gross findings were noted.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related microscopic findings were noted.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects were observed up to and including the highest dose level tested

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Dose formulation analyses: The analysed concentrations of Polyol PX were found to be between -5.8 and +2.7% of the theoretical concentration, indicating acceptable accuracy of the formulation preparation (acceptance limits ±10%). The low coefficient of variation (2.6% or lower) for the Polyol PX preparations was indicative of satisfactory homogeneity of the formulations. No test substance was detected in the control group formulations.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the NOAEL was considered to be 1000 mg/kg bw/d.

Executive summary:

A repeated dose toxicity study was conducted with Polyol PX, according to OECD 408. Polyol PX was administered daily to male and female Han Wistar rats by oral gavage for 90 days, at dose levels of 0, 100, 300 and 1000 mg/kg bw/d. The following parameters and end points were evaluated from pretreatment until study completion for all animals: viability, clinical observations, body weights, food consumption, a visual assessment of water consumption and ophthalmoscopy examinations. All animals also received a detailed functional observation battery (including motor activity) during pretreatment, Week 1, 4, 8 and Week 12/13 of treatment. Blood samples for haematology, coagulation and blood chemistry investigations and urine samples were collected from all animals during Week 13 of treatment. All surviving animals were terminated after completion of at least 91 days of treatment and underwent a detailed necropsy examination with selected organs weighed. Tissues from all control and high dose (1000 mg/kg/day) animals were subjected to a comprehensive histological examination, with gross lesions (where appropriate) examined from low and intermediate dose animals.

There was one unscheduled death; a 1000 mg/kg bw/d male was killed prematurely due to poor condition including weight loss, ploughing, subdued behaviour, reduced activity and irregular respiration. Microscopically, arteritis/periarteritis in the brain, heart and kidney, myocardial degeneration, haemorrhage in the brain, nephropathy and nephroblastoma were observed; these findings were considered spontaneous and unrelated to the administration of Polyol PX. Other than the adverse clinical observations noted for this animal, there were no treatment related clinical observations noted during treatment. There was no effect of treatment on bodyweights or food consumption profiles (with the exception of slightly lower food consumption for females that received 1000 mg/kg bw/d; body weights were unaffected) during the treatment period. There were no ophthalmoscopy findings noted during examinations following at least 91 days of treatment. There were no differences in qualitative or quantitative functional observations or motor activity assessments noted during the neurotoxicity assessment. There were no treatment-related differences in haematological, coagulation, blood chemistry or urinalysis parameters. There were no macroscopic or microscopic differences, including differences in organ weights, in treated animals noted at termination when compared with controls.

Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) was 1000 mg/kg bw/d.