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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date (Animal arrival) 26 August 2020
Experimental completion date (pathology): JUNE 15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
Purpose
The purpose of this study was to assess the influence of Hexylene glycol on reproductive performance when administered continuously by oral gavage to Sprague Dawley rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation and estrous cycles and one cohort was assessed for reproductive performance (as requested in ECHA Decision number: TPE-D-2114453322-58-01/F).



Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methylpentane-2,4-diol
EC Number:
203-489-0
EC Name:
2-methylpentane-2,4-diol
Cas Number:
107-41-5
Molecular formula:
C6H14O2
IUPAC Name:
2-methylpentane-2,4-diol
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Supplier Charles River (UK) Ltd.

Number of animals ordered 108 males and 108 females; unrelated (males not related to females).

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Six days before commencement of treatment.

Age of the F0 animals at the start of the treatment
Males: 77 to 83 days old.
Females: 70 to 76 days old.

Weight range of the F0 animals at the start of the treatment
Males: 359 to 445 g.
Females: 216 to 298 g.

Allocation to Treatment Groups (F0 Generation)
Allocation By sex, after a period of acclimatization.

Animals showing signs of ill health were excluded. Animals at the extreme of the weight range were not selected if alternatives were available.

At commencement of the study the body weight of animals did not exceed ¿20% of the mean for each sex.

Selection of Offspring to Form F1 Generation

Selection On Day 18 to 21 of age.

Allocation - formal start of F1 generation Day 21 of age (direct dose administration commenced on Day 21 of age).

Method The offspring with the lowest within-litter identification per sex from each selected litter were selected to form the F1 generation, after exclusion of grossly atypical animals.

Where possible, two males and two females were selected from each selected litter (if more were required, up to three males and three females were selected from each selected litter) and allocated to each of the two cohorts.

Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age.

Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.

Identification
Identification of animals Unique for each F0 animal and selected F1 offspring within study. All pre-weaning offspring were numbered individually within each litter on Day 1 of age.

Method Microchip (F0 generation and selected F1 generation).

Toe tattoo (pre-weaning offspring).

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24°C and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used throughout the study except during pairing.

Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.

Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Acclimatization and after selection up to four animals of one sex
Pairing one male and one female
Males to termination* up to four animals
Females after mating (from Day 0 after mating) one animal
Females during littering (from Day 20 after mating) one animal + litter
Females to termination (after weaning) up to four animals
Offspring maturation (from weaning until selection) litter
F1A animals* up to four animals of one sex
F1B animals until pairing* up to four animals of one sex

* Except when animals were separated into single housing overnight prior to urine collection )

Environmental Enrichment
Aspen wood based products A soft white untreated wood products; provided to each cage throughout the study (except for F0/F1 Cohort 1B females during lactation and when F1 Cohort 1A animals were separated into single housing overnight during urine collection) and replaced when necessary.

Plastic shelter Provided to each cage throughout the study (except for F0/F1 Cohort 1B animals when paired for mating and lactation and when F1 Cohort 1A animals were separated into single housing overnight during urine collection) and replaced at the same time as the cages .

Paper shavings From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

Diet Supply
Diet SDS VRF1 Certified, pelleted diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability Non-restricted (diet was removed overnight before blood sampling for hematology, blood chemistry and thyroid hormones and during the period of urine collection).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability Non-restricted (except during urine collection).

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen wood based products.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Purified
Details on exposure:
Method of preparation
The required amount of test item was weighed out and 50% of the final volume of purified water was added. The mixture was stirred using a magnetic stirrer. The formulation was made up to the required volume. The formulation was returned to the container and mixed using a magnetic stirrer until homogenous. It was then transferred to final containers, via syringe, whilst magnetically stirring.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation Weekly.

Storage of formulation Refrigerated (2 to 8°C).

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.



Details on mating procedure:
Mating Procedure - F0 and F1 Cohort 1B Generation
F0 pairing commenced After two weeks of treatment.

F1 Cohort 1B pairing commenced After ten weeks from selection.

Male/female ratio 1:1 from within the same treatment groups (sibling pairing was not permitted).

Duration of pairing Up to two weeks.

Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.

Day 0 of gestation When positive evidence of mating was detected.

Male/female separation Day when mating evidence was detected.

Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis
Stability and homogeneity Homogeneity and stability of the dose formulation at 5 and 200 mg/mL was established for one day at ambient temperature (15-25°C) and seven days refrigerated (2-8ºC) as part of Covance Study no. PY70VS.

Achieved concentration Samples of each formulation prepared for administration in Week 1 (F0 generation) and Week 1 and the last week of treatment (F1 generation) were analyzed for achieved concentration of the test item.

ADDITIONAL DETAILS TO BE ADDED WHEN FORMULATION ANALYSIS REPORT AVAILABLE
Duration of treatment / exposure:
Duration of Treatment
F0 animals For two weeks before pairing until termination after litters were Day 28 of age.

F1 animals From weaning until termination of respective cohort; although direct treatment started at weaning, all offspring had potential for exposure in utero and via the milk during lactation

Cohort 1A : General toxicity and pathology of the tissues of the male and female reproductive systems - treated from weaning to 13 weeks of age.

Cohort 1B : Treated from weaning throughout pairing, gestation and lactation up to weaning of F2 litters.
Frequency of treatment:
Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Details on study schedule:
Selection On Day 18 to 21 of age.

Allocation - formal start of F1 generation Day 21 of age (direct dose administration commenced on Day 21 of age).

Method The offspring with the lowest within-litter identification per sex from each selected litter were selected to form the F1 generation, after exclusion of grossly atypical animals.

Where possible, two males and two females were selected from each selected litter (if more were required, up to three males and three females were selected from each selected litter) and allocated to each of the two cohorts.

Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age.

Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
F0 generation, Group 1
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
F0 generation, Group 2
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
F0 generation, Group 3
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Remarks:
F0 generation, Group 4
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1A
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1A
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1A
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1A
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1B - control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1B
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1B
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1B
No. of animals per sex per dose:
20 males, 20 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Dose Level Selection
The doses used in this study (0, 100, 250 and 800 mg/kg/day) were selected in conjunction with the Sponsor and based on the results of a preliminary Reproduction/Developmental Toxicity Screening Test (CIT Study number 36609 RSR) and on the findings from a preliminary Pre and Post-natal Development study conducted at these laboratories (Covance Study no. WL45XP).

In the preliminary Reproduction/Developmental Toxicity Screening Test, treatment at 1000 mg/kg/day was tolerated by the dams, but there was a marked increase in pup mortality and two litters out of ten at that dose died. In the preliminary Pre and Post-natal Development study (females only), doses of 250 or 800 mg/kg/day were well-tolerated in both the F0 and the F1 generation (treated from Day 21 to Day 27 of age). There were no signs attributed to dosing or effect on clinical condition, body weight gain or food intake and there were no treatment related macroscopic findings in the F0. Gestation length, gestation index and parturition were unaffected and there was no conclusive effect on litter size or sex ratio or on the subsequent survival and growth of the offspring to weaning.

The offspring in one litter at 800 mg/kg/day showed poor growth and a relationship to treatment was uncertain. There were no signs attributed to dosing or effect on clinical condition of the treated F1 and the animals continued to grow after weaning. Food intake was unaffected, and there were no treatment related macroscopic findings.

It was therefore considered that 800 mg/kg/day was suitable for investigation as the high dose in this extended one-generation main reproductive performance study (OECD 443). The dose of 100 mg/kg/day was selected as the low dose and 250 mg/kg/day, the low dose in the preliminary study, was selected as the intermediate dose.

Examinations

Parental animals: Observations and examinations:
Serial Observations
Clinical Observations - F0 and F1 Generation
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 generation Week 1 - daily.
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onward - once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 20 of lactation for females).

F1 generation From Day 21 of age - daily.
Formal Week 1 - daily.

Weeks 2 to 4 - twice weekly (middle and end of the week)

Week 5 onward - once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 20 of lactation for females).

Detailed observations were recorded at the following times in relation to dose administration:
• Prior to dosing.
• One to two hours after completion of dosing
• As late as possible in the working day.

Detailed Physical Examination and Arena Observations
Before treatment commenced, during each week of treatment, after F1 formal selection, Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.

After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Body Weight - F0 and F1 Generation
The weight of animals was recorded as follows:

F0 males Day that treatment commenced.
Each week.
Before necropsy.

F0 females Day that treatment commenced.
Each week until mating detected.
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14, 21 and 28 of lactation.
Before necropsy

F1 selected animals Days 21, 23, 25, 27* and 29* of age.
Each week.

F1 Cohort 1B females on Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating and Days 1, 4, 7, 14, 21 and 28 post-partum.

Before necropsy.
* Only applicable before formal commencement of the F1 generation at nominal 4 Weeks of age (Day 28 of age ±2 days; not reported).

Food Consumption - F0 and F1 Generation
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 males and females Weekly, from the day that treatment commenced, until paired for mating.

Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.

For females after mating food consumption was performed to match the body weight recording:

Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18 19 after mating

Days 1-3, 4-6, 7-13, and 14-20 of lactation.(fed ad-libitum from Day 21 of lactation).

F1 selected animals Cohort 1A: Twice weekly during nominal Week 4, then weekly until termination.

Cohort 1B: Twice weekly during nominal Week 4, then weekly until paired for mating.
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18 19 after mating
Days 1-3, 4-6, 7-13, and 14-20 of lactation (fed ad-libitum from Day 21 of lactation).

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Urinalysis - F0 and F1 Cohort 1A Generation
Urine samples were collected after overnight withdrawal of food and water at the following occasions:

Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group

The individual samples were examined for the following characteristics:

• Using manual methods:
• Clarity and Color (App) - by visual assessment
• Volume (Vol) - using a measuring cylinder
• pH - using a pH meter
• Specific gravity (SG) - by direct refractometry using a SG meter

• Using Multistix reagent strips interpreted using the Clinitek®500 instrument:
• Glucose (Gluc)
• Ketones (Keto)
• Bile pigments (Bili)
• Blood pigments (UBld)

• Using a Cobas 6000 Analyzer:
• Protein - total (T-Prot) and concentration (Prot)
• Sodium - total (T-Na) and concentration (U-Na)
• Potassium - total (T-K) and concentration (U-K)
• Chloride - total (T-Cl) and concentration (U-Cl)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.

• Epithelial cells (Epi)
• Leucocytes (WBC)
• Erythrocytes (RBC)
• Casts
• Spermatozoa
• Other abnormal components (A)

Thyroid Hormone Analysis - TSH and T4
Blood samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group

F1 Offspring Ten litters per group - pooled litter sample Day 4 of age#
Ten male and ten female animals per group on Day 22 of age (from as many litters as possible)

F1 Adults - Cohort 1A Ten male and ten female animals per group (approx. 13 weeks of age)
# T4 only.

Conditions Adults: Following overnight deprivation of food.

Offspring Day 4 and Day 22: No overnight deprivation of food.
Blood sample site Adults: Sublingual vein.
Offspring Day 4 of age: Decapitation
Offspring Day 22 of age: Orbital sinus
Anaesthetic Adults and offspring on Day 22 of age: Isoflurane.
Offspring Day 4 of age: not required
Anticoagulant None.
Blood tube Greiner Minicollect - with clot activator.
Blood volume Adults and offspring Day 22 of age: 1.0 mL.
Offspring Day 4 of age: maximum possible.
Treatment of samples Samples were kept at ambient temperature (15 to 25¿C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
Serum tubes Serum was transferred to appropriately labelled polypropylene “cryo” tubes using plastic disposable pipettes.
Number of aliquots Adults and offspring Day 22 of age: Two per animal.

• Aliquot 1: 0.2 mL serum for T4
• Aliquot 2: residual serum for TSH

Offspring Day 4 of age: single aliquot.
• Maximum possible
Final storage conditions Deep frozen (approximately -60°C to -90¿C).
Fate of samples Aliquot 1 (T4): dispatched to the Department of Bioanalysis, Covance.
Aliquot 2 (TSH): dispatched to the Department of Immunology and Immunotoxicity, Covance.
T4 Performed by the Department of Bioanalysis, Covance.

Oestrous cyclicity (parental animals):
Estrous Cycle Monitoring - F0 and F1 Cohort 1B Generation
Dry and wet smears were taken as follows:

Dry smears - F0 females only For 15 days before pairing, using cotton swabs.

Wet smears - F0 and F1 Cohort 1B females After pairing until evidence of mating confirmed.

For four days before scheduled termination (nominally Days 25 to 28 post partum). Females that failed to litter were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 post partum females started smearing, and were then killed with that first batch of females.
Sperm parameters (parental animals):
Sperm Analysis - F0 and F1 Cohort 1A
Immediately after scheduled sacrifice of each F0 and F1 Cohort 1A male and collection of blood, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.

The following tests were performed:
Sperm motility: all groups A sample of sperm was expressed from the left vas deferens (with the exception of F0 males Group 1; No. 19 and Group 3; No. 51 where tissues were taken from the right side) into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and, at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyzer (CASA).

Sperm morphology: Groups 1 and 4 A 200 ¿L aliquot of the sperm/medium mixture (described above) was diluted with 800 ¿L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, with the exception of F0 male Group 1; No. 7, F1A males Group 1; No. 418 and Group 4; No. 471 where assessment of 200 sperm was unable.

Sperm morphology: Groups 2 and 3 Fixed samples retained for possible future assessment.
Sperm count: Groups 1 and 4 The left cauda epididymis (with the exception of F0 males Group 1; No. 19 and Group 3; No. 51 where tissues were taken from the right side) of each male was weighed and then the tunica was removed. The portion obtained was weighed then frozen. Prior to analysis the cauda epididymis portions were allowed to thaw then homogenized for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3 Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4 After removal of the tunica, the left testis (with the exception of F0 males Group 1; No. 19 and Group 3; No. 51 where tissues were taken from the right side) of each male was frozen. Prior to analysis, the testes were allowed to thaw then homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed.

Homogenization-resistant spermatid counts: Groups 2 and 3 Samples frozen for possible future assessment.
Litter observations:
Records Made During Littering Phase - F0 and F1 Cohort 1B Generation
The records maintained were as follows:

Clinical observations Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment.

On Day 1 of age, all offspring received a qualitative assessment of body temperature, state of activity and reaction to handling.

Litter size Daily records were maintained of mortality and consequent changes in litter size during Days 1-21 of age.

On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter.

Sex ratio of each litter Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
Individual offspring body weights Recorded on Days 1, 4 (before culling), 7, 14, 21 and 22 (day of necropsy) of age.

Weaning of offspring The dam was removed from the litter cage and offspring were weaned on Day 21 of age.

Ano-genital distance Day 1 of age - all offspring.
Nipple/areolae count Day 13 of age - male offspring.
Postmortem examinations (parental animals):
Terminal Investigations
Time of Necropsy
F0 males After weaning of the F1 animals, after confirmation that no further mating required.

F0 females Day 28 post partum.

F0 females failing to produce a viable litter Terminated with first cohort of females with live litters.

Macroscopic examination
All animals, including surplus offspring culled on Day 4 of age and Day 22 of age unselected offspring were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. Decedents (offspring
For F0 and F1 Cohort 1B females the implantation site count was recorded.

For females of F1 Cohort 1A, counts were performed for the number of ovarian follicles and corpora lutea.

Pathology procedures - F0 (Parental) Animals
Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides ¿
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Optic nerves
Ovaries ¿
Pancreas
Pituitary ¿
Prostate - dorsolateral and ventral combined ¿
Rectum
Sciatic nerves
Seminal vesicles (with coagulating gland) ¿
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum - bone marrow
Stomach
Testes ¿
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix and oviducts¿
Vagina ¿
Vas Deferens ¿

Histology
F0 animals
Wet tissues Wet tissues were dispatched to the Test Site (Covance Harrogate, UK) for processing.

Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

All procedures were performed in compliance with local Standard Operating Procedures.

Full List All adult animals killed or dying prematurely.

All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.

Processing - Males and females; liver, adrenal glands and abnormalities only

Males only kidneys All terminal adult animals of Groups 2 and 3 killed at a scheduled interval.

Processing - reproductive organs only The reproductive organs were examined from F0 animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to sire a pregnancy and females that were not pregnant.

Routine staining Sections were stained with hematoxylin and eosin.

On completion of the study phase the Histology slides were transferred back to the Test Facility with any necessary supporting documentation. The wet tissues, blocks and raw paper data were returned to the Test Facility.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified in the relevant pathology procedures table. Requisite organs were weighed for animals killed at scheduled intervals.

Light Microscopy - F0 Animals
Premature deaths F0, F1A, F1B All animals from all groups.

Scheduled kill
F0 animals All animals of Groups 1 and 4.
All animals of Groups 2 and 3. Liver, adrenal glands and abnormalities only
Males only from Groups 2 and 3 Kidney
All animals of Groups 2 and 3 with suspect fertility Reproductive organs.

Right testis A detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Ovaries F0- Qualitative evaluation of 1 section from each ovary.

Vagina The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).
Postmortem examinations (offspring):
Terminal Investigations
Time of Necropsy
F1 Cohort 1B females Day 28 post partum.

F1 Cohort 1B females failing to produce a viable litter Terminated with first cohort of females with live litters.

Unselected offspring Culled on Day 4 and Day 22 of age.

F1 Cohort 1A animals At approximately 13 weeks of age.

F1 Cohort 1B males At approximately 17 weeks of age (after weaning of F2 offspring).


Macroscopic examination
All animals, including surplus offspring culled on Day 4 of age and Day 22 of age unselected offspring were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. Decedents (offspring ¿21 days of age that were found dead or welfare kill), where possible, were examined and carcass retained.

F1 Cohort 1B females the implantation site count was recorded.

For females of F1 Cohort 1A, counts were performed for the number of ovarian follicles and corpora lutea.


Pathology procedures - F1 Adult animals (Cohort 1A)
Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
left axillary
Optic nerves
Ovaries
Pancreas
Pituitary
Prostate - dorsolateral and ventral combined
Rectum
Sciatic nerves
Seminal vesicles (with coagulating gland)
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum - bone marrow
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix and oviducts
Vagina
Vas Deferens


Pathology procedures - F1 Adult animals (Cohort 1B)
Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
left axillary
Optic nerves
Ovaries
Pancreas
Pituitary
Prostate - dorsolateral and ventral combined
Rectum
Sciatic nerves
Seminal vesicles (with coagulation gland)
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum - bone marrow
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix and oviducts
Vagina
Vas Deferens

Pathology procedures - Unselected F1 and F2 offspring (Day 22 of age)
Ten male and ten females per group; one male or one female from each litter to ensure that all litters are represented

Tissue and regions examined

Abnormalities
Brain (cerebellum, cerebrum, midbrain)
Epididymides
Ovaries
Pituitary
Prostate
Seminal vesicles
Skin with mammary glands (inguinal area)
Spleen
Testes
Thymus
Uterus with cervix and oviducts
Vagina

PLEASE REFER TO SECTION ABOVE "SPERM PARAMETERS - PARENTAL ANIMALS" RE SPERM PARAMETRS FOR OFFSPRING

Histology
F0 animals and F1 Cohort 1A
Wet tissues Wet tissues were dispatched to the Test Site (Covance Harrogate, UK) for processing.

Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

All procedures were performed in compliance with local Standard Operating Procedures.

Full List All adult animals killed or dying prematurely.

All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.

Processing - Males and females; liver, adrenal glands and abnormalities only

Males only kidneys All terminal adult animals of Groups 2 and 3 killed at a scheduled interval.

Processing - reproductive organs only The reproductive organs were examined from F0 animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to sire a pregnancy and females that were not pregnant.

Routine staining Sections were stained with hematoxylin and eosin.

On completion of the study phase the Histology slides were transferred back to the Test Facility with any necessary supporting documentation. The wet tissues, blocks and raw paper data were returned to the Test Facility.

F1 Cohort 1B
Processing Tissue samples were dehydrated and embedded in paraffin wax.

Limited list (to block) All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.
Abnormalities (to block) All terminal adult animals of Groups 2 and 3 killed at a scheduled interval.

Immunophenotyping of Spleen Leucocytes - F1 Cohort 1A
Ten males and ten females per group from F1 Cohort 1A were selected for immunophenotyping.

Where possible, one male or one female was assigned from each selected litter.

The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological evaluation. The remaining portions of the spleen was then weighed, placed in to a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held in wet ice until processing for analysis.

Samples were sent via courier to Department of Immunology and Immunotoxicology (I&I), Covance. A copy of the whole spleen and partial spleen weights were provided to I&I.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified in the relevant pathology procedures table. Requisite organs were weighed for animals killed at scheduled intervals.

For unselected F1 offspring on Day 22 of age, organs were weighed from ten males and ten females per sex per group from as many litters as possible.

Immunophenotyping of Spleen Leucocytes - F1 Cohort 1A
Ten males and ten females per group from F1 Cohort 1A were selected for immunophenotyping. Where possible, one male or one female was assigned from each selected litter.

The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological evaluation. The remaining portions of the spleen was then weighed, placed in to a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held in wet ice until processing for analysis.

Samples were sent via courier to Department of Immunology and Immunotoxicology (I&I), Covance. A copy of the whole spleen and partial spleen weights were provided to I&I.

F1 Cohort 1A
Tissues preserved for examination were examined as follows:
Premature deaths F0, F1A, F1B All animals from all groups.

F1A All animals of Groups 1 and 4. All specified in Table 3
All animals of Groups 2 and 3. Liver, adrenal glands and abnormalities only
Males only from Groups 2 and 3 Kidney
F1B All animals. Abnormalities only.

Right testis A detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Ovaries F0/F1A - Qualitative evaluation of 1 section from each ovary.
F1 Cohort 1A - Quantitative evaluation of 5 sections from the middle third from each ovary for the assessment of primordial follicle and small growing follicle populations as well as evaluation of corpora lutea numbers in one section from each ovary.

Vagina The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).
Statistics:
Data Evaluation
This report contains serial observations pertaining to all days or weeks of treatment completed, together with signs data collected during the necropsy period. No serial observations relating to the acclimatization period are included in this report.

Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data. The summary statistics and the individual data were stored in the computer to a certain number of decimal places, different for each parameter. For presentation purposes, however, they were usually rounded to fewer places. It is, therefore, not generally possible to reproduce the presented means and standard deviations exactly using the presented individual data.

Throughout the report the following abbreviations were used:
M Male
F Female
SD Standard deviation
N Number contributing to the mean (normally the number of animals/litters)

PLEASE REFER TO ANY OTHER INFORMATION ON MATERIALS AND METHODS"
Reproductive indices:
Pre-coital Interval - F0 and F1 Cohort 1B Generation
Individual intervals were tabulated for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 or 13 14 days of pairing.

Mating Performance and Fertility - F0 and F1 Cohort 1B Generation
Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating/ Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy /Animals paired) x 100

Gestation Length and Gestation Index - F0 and F1 Cohort 1B Generation
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.

Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born / Animals paired) x 100
Offspring viability indices:
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after culling), 7, 14 and 21 of age. Group mean litter size and SD were calculated from the individual litter values.

Survival Indices - F0 and F1 Cohort 1B Generation
The following were calculated for each litter:

Post implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 before culling / Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 21 after littering / Number of live offspring on Day 4 (after culling)) x 100

Group mean values were calculated from individual litter values.
Sex Ratio - F0 and F1 Cohort 1B Generation

The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after culling) and 21 of age.

Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Group mean values were calculated from individual litter values.

Offspring Examinations - F0 and F1 Cohort 1B Generation
Ano-genital distance were presented both as absolute/unadjusted and adjusted for body weight, using the weight recorded on Day 1 of age.

A check was performed to assess for the presence or absence of nipple/areolae for the male offspring on Day 13 of age. As no nipples were present for F1 males, no data are included.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical Observations
There were no signs attributed to oral gavage administration in males, or in non-mated females during the two-week pre-pairing period, or females during gestation or lactation.

All clinical signs observed during the detailed physical examination and arena observations, showed no relationship to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were four decedents in the F0 generation, none were attributed to treatment with Hexylene glycol.

One male (No. 46) receiving 100 mg/kg/day that was previously clinically normal was found dead shortly after dosing on Study Day 54. Macroscopically, perforation and distension of the esophagus were present along with fluid, adhesions and abnormal contents in the thoracic cavity. Histopathology indicated inflammation and abscess formation in the thoracic cavity that were consistent with mis-dosing were apparent and this was the cause of death.

One male (No. 55) receiving 250 mg/kg/day was killed for welfare reasons on Study Day 36. Trauma wounds, including bruising, depressions and eschar formation were apparent. Macroscopically, discoloration and a depression with an open area were present on the skin. The major microscopic change was abscess formation in the skin/subcutaneous tissue and this was considered to be the cause of the poor clinical condition and therefore the cause of death.

One male (No. 78) receiving 800 mg/kg/day that was previously clinically normal was killed for welfare reasons on Study Day 54, due to signs of gasping respiration and decreased activity. Macroscopic perforation of the esophagus was consistent with mis-dosing and inflammatory change in the lungs correlated with this.

One male (No. 90) receiving 800 mg/kg/day that was previously clinically normal was found dead shortly after dosing on Study Day 36. Macroscopically, pale areas were present in the lungs and the glandular mucosa in the stomach was thickened. The major histopathology change was chronic, active inflammation in the heart and this was considered to be the cause of death. The aetiology of this was not clear but it was considered not to be related to treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall body weight gain at 100, 250 or 800 mg/kg/day was unaffected by treatment during the two-week pre-pairing period for males and females or until scheduled necropsy of the males.

Overall body weight gain was unaffected by treatment during gestation at 100, 250 or 800 mg/kg/day.

Overall body weight gain was unaffected by treatment during lactation at 100, 250 or 800 mg/kg/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption at 100, 250 or 800 mg/kg/day was unaffected by treatment during the two week pre-pairing period for males and females or until scheduled necropsy of the males.

Overall food consumption was unaffected by treatment during gestation at 100, 250 or 800 mg/kg/day.

Overall food consumption was unaffected by treatment during lactation at 100, 250 or 800 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant differences in the cellular composition of the blood in Week 10 of treatment in males and on LD 28 in females. All inter-group differences were within the 95-percentile of the HCD range, unless indicated.
In males at 800 mg/kg/day and in females at 100, 250 or 800 mg/kg/day and without relationship to treatment, prothrombin time was short (88%* and 86%*, 73*%% or 83%** of Control, respectively).
In males at 800 mg/kg/day, neutrophil count was high (166%** of Control) that exceeded the HCD range; however, as this was seen in one sex and with no microscopic correlate, it was considered not to be toxicologically important. Monocyte count was high (155%* of Control) and platelet count was marginally high (116%* of Control) in males at 800 mg/kg/day. In females at 800 mg/kg/day, hematocrit and red cell count were marginally low (96%** and 95%*, respectively) and mean cell hemoglobin and mean cell hemoglobin concentrations were marginally high (104%* and 103%* respectively).


PLEASE REFER TO THE ATTACHED TABLE: "HAEMATOLOGY - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F0)"
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant differences in the constituents of the blood plasma in Week 10 of treatment in males and on LD 28 in females. All inter-group differences were within the 95-percentile of the HCD range, unless indicated.
In males and females at 800 mg/kg/day, glucose concentration was low (88%** and 86%* of Control, respectively) that exceeded the HCD range in males only and at 250 or 800 mg/kg/day, total protein concentrations were high (106%** or 114%** and 105% or 105%*, respectively) that exceeded the HCD range in males only at 800 mg/kg/day and correlated with marginally high albumin concentration (108%) that also exceeded the HCD range and low albumin globulin ratio at 100, 250 or 800 mg/kg/day (92%*, 91%** or 88%**, respectively). The changes in total protein and albumin concentrations in males only were attributed to the microscopic changes identified in the liver.
In males only at 250 or 800 mg/kg/day, alkaline phosphatase activities were increased (121%* or 138%** of Control, respectively) and at 800 mg/kg/day, alanine amino transferase activity was 200%** of Control. Aspartate amino transferase activity was 174%** of Control and exceeded the HCD range. Bilirubin, bile acid (no HCD available) and cholesterol concentrations were high (200%**, 264%* and 195%**, respectively) and creatinine concentration was low (90%*).


PLEASE REFER TO THE ATTACHED TABLE: "BLOOD CHEMISTRY - GROUP MEAN VALUES AT SCHEDULED TERMNINATION (F0)"
Endocrine findings:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis - TSH and T4
Serum TSH and T4 concentrations at termination in F0 and F1 adults and F1 offspring on Day 22 of age were considered to be unaffected by treatment at 100, 250 or 800 mg/kg/day. There was variation in the group mean results and mean serum T4 concentration was statistically significantly low in F1 males treated at 800 mg/kg/day; however, female concentrations were unaffected at any dose.

PLEASE REFER TO TABLES IN "ANY OTHER INFORMATION ON RESULTS": "GROUP MEAN SERUM TSH CONCENTRATION DATA IN MALE RATS"
and
"GROUP MEAN SERUM TSH CONCENTRATION DATA IN FEMALE RATS"
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant differences in the composition of the urine in Week 10 of treatment in males and on LD 28 in females.

Urinary pH was marginally low in males at 250 or 800 mg/kg/day and in females at 800 mg/kg/day (91%* or 79%** and 83%**, respectively). Specific gravity was marginally high in males and females at 800 mg/kg/day (both 101%**).

In males only, total sodium concentration was high at 800 mg/kg/day (171%**) and correlated with a high volume of urine in the same animals (140%*).

PLEASE REFER TO THE ATTACHED TABLE: "URINALYSIS - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F0)"
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to the administration of Hexylene glycol were seen in the adrenal glands and liver of males and females and the kidneys of males.
Diffuse cortical hypertrophy, minimal or slight, was present in the adrenal gland of males and females given 800 mg/kg/day. Vacuolation of the cortex was increased in males given all doses of Hexylene glycol. The hypertrophy, which correlated with the changes in organ weight, was considered to be treatment related.
Centrilobular hypertrophy was present in the liver of males and females given 800 mg/kg/day. Bile duct hyperplasia was present in males given 800 mg/kg/day.
In the kidneys of males given all doses of Hexylene glycol there was an increase in the incidence and severity of hyaline droplet accumulation in the tubular cells. Basophilic tubules were increased in those givem 100 or 250 mg/kg/day but this showed no relation to treatment and was not apparent in those given 800 mg/kg/day thus is considered unrelated to treatment. This is considered to be a specific effect to male rats and is not relevant to humans.


PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "INCIDENCE AND SEVERITY OF HEXYLENE-GLYCOL RELATED MICROSCOPIC FINDINGS - TERMINAL SACRIFICE (F0)
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycles at pairing were unaffected by treatment at 100, 250 or 800 mg/kg/day.

All females receiving 100, 250 or 800 mg/kg/day showed estrus following lactation, during the four day period immediately before termination (LD 25-28).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
When compared with Control, it was considered that there was no adverse effect on sperm motility, morphology or testicular or cauda epididymis sperm counts at 100, 250 or 800 mg/kg/day.
Reproductive performance:
no effects observed
Description (incidence and severity):
Pre-Coital Interval
Pre-coital interval was unaffected by treatment at 100, 250 or 800 mg/kg/day.

Mating Performance and Fertility
Mating performance and fertility were unaffected by treatment at 100, 250 or 800 mg/kg/day. One pairing from the Control group (Female No. 216) failed to result in pregnancy, following evidence of a successful mating. Macroscopic examination of the female revealed no corpora lutea or implantation sites and there were no microscopic present to account for this.

Gestation Length and Gestation Index
Gestation length was considered to be unaffected by treatment at 100, 250 or 800 mg/kg/day, as all animals littered within 22-23.5 days of mating

Gestation index was unaffected by treatment.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: All endpoints examined
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs attributed to oral gavage administration in males, or in non-mated females or females during gestation or lactation.
All clinical signs observed during the detailed physical examination and arena observations, showed no relationship to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male (No. 500) from the Control group that was previously clinically normal was found dead shortly after dosing on Study Day 79; there were no macroscopic or microscopic findings and the cause of death was undetermined.
One male (No. 521) receiving 250 mg/kg/day showed decreased activity and impaired respiration (irregular and shallow respiration) and was killed for welfare reasons on Study Day 100. Macroscopically, there was perforation of the esophagus and abnormal contents/adhesions in the thoracic cavity. Microscopically inflammation of the pleura was present and death was caused by a dosing accident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 800 mg/kg bw/day, absolute body weight was transiently, but significantly low on Days 1, 8 and 15 in males and on Days 1 and 8 in females and this was considered to be treatment related.
Overall body weight gain at 100, 250 or 800 mg/kg/day was unaffected by treatment in males and in un-mated females from weaning to scheduled termination (Cohort 1A; 13 weeks of age; Cohort 1B after weaning of the F2 litters).
Mean absolute body weight of Cohort 1B females at 800 mg/kg/day was marginally low (93%**) on GD 0, when compared with Control. This was considered to be treatment related.
Investigation showed lower than Control body weight gains at 100, 250 or 800 mg/kg/day during the period of pairing. The reason for the low weight gain in treated animals during pairing was not known, but was considered not to be toxicologically significant.
Absolute body weight at 800 mg/kg/day remained low, when compared with Control, to Day 18 of gestation; however, overall body weight gain at 100, 250 or 800 mg/kg/day was unaffected by treatment.
Absolute body weight on LD 1 at 800 mg/kg/day was marginally, but statistically significantly low (95%* of Control); the difference was considered not to be toxicologically significant. Overall body weight gain (LD 1-21) at 100, 250 or 800 mg/kg/day was superior to that of the Control (2.3-3.3-fold**).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption at 100, 250 or 800 mg/kg/day was unaffected in males and females during the 10-week pre pairing period.
Overall food consumption was unaffected by treatment during gestation at 100, 250 or 800 mg/kg/day.
Overall food consumption was unaffected by treatment during lactation at 100, 250 or 800 mg/kg/day.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Description (incidence and severity):
see cohort 1 A below
Clinical biochemistry findings:
not examined
Description (incidence and severity):
see cohort 1 A below
Urinalysis findings:
not examined
Description (incidence and severity):
see cohort 1A below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Reproductive organs and thymus weights were unaffected by treatment in Cohort 1B.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for this animal age and/or strain and age. Consequently, they were considered not test article related.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
adaptative effects seen in the adrenals, the liver ,
non specific effects in the males kidneys
see dicussion below

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycles from Day 75 of age were considered unaffected by treatment at 100, 250 or 800 mg/kg/day.
One female at 100 mg/kg/day was acyclic (at least ten days without estrus), one female at 250 mg/kg/day had an irregular cycle (at least one cycle of two, three or six to ten days) and two females were acyclic and one female had an irregular cycle at 800 mg/kg/day that were within the HCD range and therefore considered not to be toxicologically significant (see Attachment 14.8).
Reproductive performance:
no effects observed

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Offspring remained in good clinical condition until scheduled termination on PND 22. Commonly seen signs of attached umbilical cord, little or no milk in stomach, bruising, cuts and patchy hair growth were seen at low incidence and showed no relationship to parental treatment at 100, 250 or 800 mg/kg/day.

There were no signs attributed to oral gavage administration in males, or in non-mated females or females during gestation or lactation.
All clinical signs observed during the detailed physical examination and arena observations, showed no relationship to treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
One male (No. 500) from the Control group that was previously clinically normal was found dead shortly after dosing on Study Day 79; there were no macroscopic or microscopic findings and the cause of death was undetermined.
One male (No. 521) receiving 250 mg/kg/day showed decreased activity and impaired respiration (irregular and shallow respiration) and was killed for welfare reasons on Study Day 100. Macroscopically, there was perforation of the oesophagus and abnormal contents/adhesions in the thoracic cavity. Microscopically inflammation of the pleura was present and death was caused by a dosing accident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall body weight gain PND 1 to PND 21 was unaffected by parental treatment at 100, 250 or 800 mg/kg/day.

The absolute body weight of male offspring on PND 1 was marginally, but statistically significantly high (107% of Control) at 800 mg/kg/day.

Overall body weight gain at 100, 250 or 800 mg/kg/day was unaffected by treatment in males and in un-mated females from weaning to scheduled termination (Cohort 1A; 13 weeks of age; Cohort 1B after weaning of the F2 litters).
Mean absolute body weight of Cohort 1B females at 800 mg/kg/day was marginally low (93%**) on GD 0, when compared with Control. Investigation showed lower than Control body weight
gains at 100, 250 or 800 mg/kg/day during the period of pairing. The reason for the low weight gain in treated animals during pairing was not known, but was considered not to be toxicologically significant.

Overall body weight gain during gestation at 100, 250 or 800 mg/kg/day was unaffected by treatment.
Absolute body weight on LD 1 at 800 mg/kg/day was marginally, but statistically significantly low (95%* of Control); the difference was considered not to be toxicologically significant. Overall body weight gain (LD 1-LD 21) at 100, 250 or 800 mg/kg/day was superior to that of the Control (2.3-3.3-fold**).


PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "BODY WEIGHT (G) - GROUP MEAN VALUES OF FEMALES BEFORE PAIRING AND ON GD 0"
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption at 100, 250 or 800 mg/kg/day was unaffected in males and females during the 10-week pre pairing period.
Overall food consumption was unaffected by treatment during gestation at 100, 250 or 800 mg/kg/day.
Overall food consumption was unaffected by treatment during lactation at 100, 250 or 800 mg/kg/day.
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):
Cohort 1A
There were no toxicologically significant differences in the cellular composition of the blood following 10 weeks of direct treatment (approximately Week 13 of age).

Platelet count was high in males treated at 800 mg/kg/day (115%** of Control).
In females at 250 or 800 mg/kg/day, reticulocyte count was slightly low, without relationship to dose (81%* or 83%** of Control, respectively). Neutrophil count was low in females treated at 800 mg/kg/day (63%*) and eosinophil count was low in males at 800 mg/kg/day (58%*) and females at 100, 250 or 800 mg/kg/day (62%*, 77%* or 46%** of Control, respectively); however, overall white cell counts were unaffected.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Blood Chemistry - Cohort 1A
There were no toxicologically significant differences in the constituents of the blood plasma following 10 weeks of direct treatment (approximately Week 13 of age).

Cholesterol concentration was high in males and females at 800 mg/kg/day (132%** or 125%* of Control, respectively). Total plasma protein concentrations were marginally high in males and females at 800 mg/kg/day (108%** or 106%*, respectively) and correlated in males only with marginally high albumin concentration at 250 or 800 mg/kg/day (both 106%*).
In males only, alanine amino-transferase activity was high without relationship to dose at 250 or 800 mg/kg/day (145% or 148% of Control, respectively). Bile acid concentration was markedly high in females treated at 800 mg/kg/day (7.5-fold**); however, only the values for two animals exceeded the Control group range; therefore no relationship to treatment is inferred. Glucose concentration was slightly low in males at 250 or 800 mg/kg/day (87%* or 80%*, respectively). Potassium concentration was marginally high in females at 250 or 800 mg/kg/day (107* or 111%**) and calcium concentration was marginally high in males at 800 mg/kg/day (105%**).

PLEASE REFER TO THE ATTACHED TABLE: "BLOOD CHEMISTRY - GROUP MEAN VALUES AT SCHEULED TERMINATION (F1 COHORT 1A)"
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A
There were no toxicologically significant differences in the composition of the urine following 10 weeks of direct treatment (approximately Week 13 of age).

Urinary pH was low in males and females at 250 or 800 mg/kg/day (88%** or 81%* and 94%* or 91%* of Control, respectively) and specific gravity was marginally high in males only at these doses (101%* or 102%*, respectively). In females only, total protein and potassium concentrations were high at 800 mg/kg/day (both 142%* of Control).

PLEASE REFER TO THE ATTACHED TABLE: "URINALAYSIS - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F1 COHORT 1a)"
Sexual maturation:
no effects observed
Description (incidence and severity):
All animals at 100, 250 or 800 mg/kg/day showed sexual maturation within one day of Control and was therefore unaffected by treatment.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Ano-genital distance on Day 1 of age was unaffected by parental treatment at 100, 250 or 800 mg/kg/day.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
A check was performed to assess for the presence or absence of nipple/areolae for the male offspring on Day 13 of age. As no nipples were present for F1 males, no data are included.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no signs attributed to oral gavage administration in males, or in non-mated females or females during gestation or lactation.

All clinical signs observed during the detailed physical examination and arena observations, showed no relationship to treatment.

Body weight relative brain weight was marginally low in females following parental treatment at 800 mg/kg/day (91%*).

F1 generation Cohort 1A
There was an increase in absolute and body weight relative weight of the adrenal glands of males given 800 mg/kg and females given 250 and 800 mg/kg/day. There was an increase in absolute and body weight relative weight of the kidneys of males given 800 mg/kg/day and females given 250 and 800 mg/kg/day, in addition the body weight relative weight only was increased in males given 100 and 250 mg/kg/day. There was an increase in absolute and body weight relative weight of the liver of males and females given 800 mg/kg/day. There was an increase in body weight relative weight of the prostate of males given 800 mg/kg/day and seminal vesicle (including coagulating glands) of males given all doses and the absolute and body weight relative weight of the ovaries of females given all doses.

All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.
Reproductive organs and thymus weights were unaffected by treatment in Cohort 1B.


PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "HEXYLENE GLYCOL-RELATED EFFECTS IN ORGAN WEIGHTS PARAMETERS - TERMINAL SACRIFICE (F1)"
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic findings in the F1 offspring on PND 22 were considered to be unrelated to parental treatment at 100, 250 or 800 mg/kg/day.

F1 generation Cohort 1A and 1B
No macroscopic changes were seen related to the administration of Hexylene Glycol.
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including Control), and/or were as expected for this animal age and/or strain and age. Consequently, they were considered not test article related.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Histopathology - Cohort 1A
Changes related to the administration of Hexylene glycol were seen in the adrenal glands and liver of males and females and the kidneys of males.
Increased diffuse cortical hypertrophy, minimal, was present in the adrenal gland of males and females given 800 mg/kg/day. Vacuolation of the cortex was increased in males given 800 mg/kg/day and marginally in those given 250 mg/kg/day.
Centrilobular hypertrophy was present in the liver of males and females given 800 mg/kg/day. Bile duct hyperplasia was not increased in F1 males.
In the kidneys of males given all doses of Hexylene glycol there was an increase in the incidence and severity of hyaline droplet accumulation in the tubular cells. In addition there was an increase in incidence of basophilic tubules, with multifocal changes at minimal or slight severity in males given 250 or 800 mg/kg/day. Granular casts were also present in one male given 250 and three males given 800 mg/kg/day.


PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "INCIDENCE AND SEVERITY OF HEXYLENE-GLYCOL RELATED MICROSCOPIC FINDINGS - TERMINAL SACRIFICE (F1)"
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis - TSH and T4
Serum TSH and T4 concentrations at termination in F0 and F1 adults and F1 offspring on Day 22 of age were considered to be unaffected by treatment at 100, 250 or 800 mg/kg/day. There was variation in the group mean results and mean serum T4 concentration was statistically significantly low in F1 males treated at 800 mg/kg/day; however, female concentrations were unaffected at any dose.

PLEASE REFER TO THE TABLES IN "ANY OTHER INFORMATION ON RESULTS": "MEAN SERUM T4 CONCENTRATIONS IN F1 OFFSPRING"
and
"MEAN SERUM T4 CONCENTRATIONS IN F0 AND F1 ANIMALS"

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Litter Size, Survival Indices and Sex Ratio
The numbers of implantations and total offspring born were unaffected by treatment at 100, 250 or 800 mg/kg/day.

A higher number of litters at 800 mg/kg/day showed offspring deaths following parturition to Day 1 of age, when compared with Control (14 v 5* litters) and was higher than the HCD range (See Attachment 14.8); live birth index was marginally low (91.2%* of Control) and also exceeded the Historical Control Data (HCD) range. Subsequently, litter size on Day 1 and on Day 4 (before culling) at 800 mg/kg/day was slightly, but statistically significantly low (90%* of Control), this was considered to be treatment related. Subsequent survival of the offspring to weaning was unaffected.
Offspring sex ratio was unaffected by treatment at 100, 250 or 800 mg/kg/day


Vaginal Opening - Cohort 1A
The interval between vaginal opening and first estrus was considered to be unaffected by treatment at 100, 250 or 800 mg/kg/day.

Estrous Cycles - Cohort 1A
Estrous cycles from Day 75 of age were considered unaffected by treatment at 100, 250 or 800 mg/kg/day.

One female at 100 mg/kg/day was acyclic (at least ten days without estrus), one female at 250 mg/kg/day had an irregular cycle (at least one cycle of two, three or six to ten days) and two females were acyclic and one female had an irregular cycle at 800 mg/kg/day that were within the HCD range and therefore considered not to be toxicologically significant.

Pre-Coital Interval - Cohort 1B
Pre-coital interval was unaffected by treatment at 100, 250 or 800 mg/kg/day.

All pairs showed evidence of successful mating within the 14 day period, as seen in the Control.

Mating Performance and Fertility - Cohort 1B
Following successful evidence of mating, four pairings at 100 mg/kg/day (Female No’s 709, 717, 718 and 720) and two pairings at 800 mg/kg/day (Female No’s 747 and 754) failed to result in pregnancy. Macroscopic examination of the females revealed no corpora lutea or implantations; therefore the animals had not been pregnant. No changes were present microscopically to account for the infertility and no significant findings were present As there was no relationship to treatment and only a small number of animals affected, no relationship with treatment was inferred.

Gestation Length and Gestation Index - Cohort 1B
Gestation length was considered to be unaffected by treatment at 100, 250 or 800 mg/kg/day, as all animals littered within 22-23.5 days of mating (See HCD).

Gestation index was unaffected by treatment.

Stage of Estrous Cycle at Termination - Cohort 1B
Estrous cycles during the four day period before termination (LD 25-28) were unaffected by treatment at 100, 250 or 800 mg/kg/day.

All females receiving 100, 250 or 800 mg/kg/day showed estrus following lactation, during LD 25-28, with the exception of one female receiving 100 mg/kg/day. This was considered not to be toxicologically significant.

Ovarian Follicle Counts and Corpora Lutea - Cohort 1A
The numbers of ovarian follicles and corpora lutea was unaffected at 100, 250 or 800 mg/kg/day.

Sperm Assessment - Cohort 1A
When compared with Control, it was considered that there was no adverse effect on sperm motility, morphology or testicular or cauda epididymis sperm counts at 100, 250 or 800 mg/kg/day.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
General Condition of Offspring
Offspring remained in good clinical condition until scheduled termination on PND 22. Commonly seen signs of attached umbilical cord, bruising and patchy hair growth were seen at low incidence and showed no relationship to parental treatment at 100, 250 or 800 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Absolute body weight of male and female offspring on PND 1 was unaffected by treatment at 100, 250 or 800 mg/kg/day; subsequent body weight gain (Days 1-21 of age) was marginally, but statistically significantly low following parental treatment at 800 mg/kg/day (both 94%* of Control).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Ano-genital distance on Day 1 of age was unaffected by parental treatment at 100, 250 or 800 mg/kg/day.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
One male offspring, following parental treatment at 100 mg/kg/day, had 1 nipple. No other offspring at 100 mg/kg/day and no offspring at 250 or 800 mg/kg/day had nipples.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights in the F2 offspring on PND 22 were unaffected following parental treatment at 100, 250 or 800 mg/kg/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic findings in the F2 offspring on PND 22 were considered to be unrelated to parental treatment at 100, 250 or 800 mg/kg/day.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Litter Size, Survival Indices and Sex Ratio
The numbers of implantations and total offspring born were unaffected by parental treatment at 100, 250 or 800 mg/kg/day.
A higher number of litters at 800 mg/kg/day showed offspring deaths during Days 1 to 4 of age (Viability Index (%), when compared with Control (7 v 1** litters) that was outside the HCD range, thus the viability index was decreased and as a consequence live litter size was marginally low at the same dose; these changes were considered to be a treatment related affect. Subsequent survival to weaning was unaffected.
The viability indices were unaffected at 100 or 250 mg/kg/day.
The ratio of male to female offspring was unaffected by parental treatment at 100, 250 or 800 mg/kg/day.


PLEASE REFER TO THE ATTACHED TABLE: "LITTER SIZE - GROUP MEAN VALUES (F1)"

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes

Any other information on results incl. tables

Formulation Analysis


The mean concentrations of Hexylene Glycol in dose formulations were within the nominal limits (-10/-15%), confirming the accuracy of formulations. The difference from mean remained within 4%, confirming precise analysis.


Analyzed Concentrations for Hexylene Glycol in Purified Water








































































































































Occasion



Group



Nominal concentration (mg/mL)



Analyzed concentration


(mg/mL)



RME (%)



Difference from mean (%)



Analysis 1



Analysis 2



Mean



Week 1 (F0 generation)



1



0



ND



ND



-



-



-



2



20



18.2



18.2



18.2



-9.0



±0.12



3



50



49.1



46.4



47.7



-4.6



±2.77



4



160



152



141



146



-8.8



±3.54



 



Week 1 (F1 generation)



1



0



ND



ND



-



-



-



2



20



19.9



19.9



19.9



-0.5



±0.14



3



50



50.4



50.5



50.5



+1.0



±0.10



4



160



162



162



162



+1.3



±0.09



 



Last week (F1 generation)



1



0



ND



ND



-



-



-



2



20



20.3



20.8



20.5



+2.5



±1.18



3



50



51.9



51.9



51.9



+3.8



±0.03



4



160



167



167



167



+4.4



±0.28



RME      Relative mean error, representing the deviation from nominal


ND          Not detected


Mean analyzed concentrations and difference from mean values were calculated using unrounded figures.


 


: Group Mean Serum TSH Concentration Data (pg/mL) in Male Rats















































































































































Group



Treatment



Dose (mg/kg/day)



 



F0 Adults
Termination



F1 Offspring Day 22
Termination



F1 Adults Cohort A
Week 13
Termination


 
 

1



Control



0



Mean



723



532



1410


 

SD



499



203



811


 

%CV



69.1



38.2



57.7


 

N



10



10



10


 

2



Hexylene
Glycol



100



Mean



1120



454



972


 

SD



499



263



379


 

%CV



44.6



57.9



39.0


 

N



10



10



10


 

3



Hexylene
Glycol



250



Mean



935



636



862


 

SD



540



333



295


 

%CV



57.7



52.4



34.2


 

N



10



10



10


 

4



Hexylene
Glycol



800



Mean



914



539



764*


 

SD



428



NA



687


 

%CV



46.8



NA



89.8


 

N



10



10



10


 

*              p<0.05


Text table 3: Group Mean Serum TSH Concentration Data (pg/mL) in Female Rats















































































































































Group



Treatment



Dose (mg/kg/day)



 



F0 Adults


Day 28 of Lactation
Termination



F1 Offspring Day 22
Termination



F1 Adults Cohort A
Week 13
Termination


 
 

1



Control



0



Mean



489



611



596


 

SD



239



378



320


 

%CV



48.8



61.9



53.6


 

N



10



10



10


 

2



Hexylene
Glycol



100



Mean



526



315



646


 

SD



265



108



245


 

%CV



50.4



34.3



37.8


 

N



10



10



10


 

3



Hexylene
Glycol



250



Mean



697



486



602


 

SD



326



231



372


 

%CV



46.7



47.5



61.7


 

N



10



10



10


 

4



Hexylene
Glycol



800



Mean



803*



561



658


 

SD



336



294



206


 

%CV



41.8



52.3



31.4


 

N



10



10



10


 

*              p<0.05


 


 


: Mean serum T4 concentrations (pg/mL) in F1 offspring










































































































Group



Treatment



Dose


 


(mg/kg/day)



Parameters



F1 offspring on Day 22 of age


(Male)



F1 offspring on Day 22 of age


(Female)



1



Control



0



Mean



36900



35800



SD



10300



9720



CV%



27.9



27.2



N



10



9



2


 



Hexylene glycol



100



Mean



40800



35300



SD



4950



6380



CV%



12.1



18.1



N



10



10



3



Hexylene glycol



250



Mean



39000



33600



SD



5200



5180



CV%



13.3



15.4



N



10



10



4



Hexylene glycol



800



Mean



40200



36200



SD



6670



5970



CV%



16.6



16.5



N



10



10



 


Text table 5: Mean serum T4 concentrations (pg/mL) in F0 and F1 adults


 












































































































































Group



Treatment



Dose


 


(mg/kg/day)



Parameters



F0 Adult Terminal (Male)



F0 Adult Terminal (Female)



F1 Adult Terminal (Male)



F1 Adult Terminal (Female)


 



1



Control


(Vehicle)



0



Mean



54800



30200



58000



39600



SD



16700



8340



9620



9890



CV%



30.5



27.6



16.6



25.0



N



10



10



10



10



2



Hexylene glycol



100



Mean



67300



33000



64200



49500



SD



8660



6360



13800



12700



CV%



12.9



19.3



21.5



25.7



N



10



10



10



10



3



Hexylene glycol



250



Mean



59200



35700



59000



44700



SD



15700



7860



6990



10000



CV%



26.5



22.0



11.8



22.4



N



10



10



10



10



4



Hexylene glycol



800



Mean



48300



34500



48900 *



45600



SD



4790



7680



6730



9910



CV%



9.9



22.3



13.8



21.7



N



10



10



10



10



*              p<0.05


 


Hexylene glycol-Related Effects in Organ Weight Parameters - Terminal Sacrifice (F0)

































































































































































































































 



Hexylene glycol



 



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



250



800



0



100



250



800



Adrenal Glands



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.060



0.062



0.062



0.077**



0.067



0.072



0.075**



0.081**



Body Weight Ratio (%)



0.011



0.012



0.012



0.014**



0.023



0.025



0.026*



0.027**



Kidneys



 



 



 



 



 



 



 



 



Absolute Weight (g)



3.67



3.79



3.83



4.33**



2.15



2.24



2.29*



2.46**



Body Weight Ratio (%)



0.67



0.71*



0.73*



0.80**



0.75



0.77



0.79*



0.83**



Liver



 



 



 



 



 



 



 



 



Absolute Weight (g)



17.5



16.8



17.5



25.7**



11.7



11.8



12.5



13.9*



Body Weight Ratio (%)



3.21



3.14



3.34



4.39**



4.09



4.06



4.28



4.71*



Spleen



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.77



0.77



0,80



0.86*



0.58



0.61



0.62



0.56



Body Weight Ratio (%)



0.14



0.14



0.15



0.16**



0.20



0.21



0.21



0.19



Thymus



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.25



0.24



0.23



0.30*



0.28



0.28



0.29



0.29



Body Weight Ratio (%)



0.05



0.04



0.04



0.06*



0.10



0.10



0.10



0.10



Ovaries



 



 



 



 



 



 



 



 



Absolute Weight (g)



 



 



 



 



0.11



0.12



0.13*



0.13*



Body Weight Ratio (%)



 



 



 



 



0.039



0.040



0.043*



0.043*



 


 


Incidence and Severity of Hexylene glycol-Related Microscopic Findings - Terminal Sacrifice (F0)






















































































































































































































































































Sex



Hexylene glycol



Males



Females



Dose Level (mg/kg/day)



0



100



250



800



0



100



250



800



Adrenal Glands



 



 



 



 



 



 



 



 



Number Examined



25



0



0



23



24



0



0



25



 Hypertrophy, Cortical, Diffuse



 



 



 



 



 



 



 



 



Minimal



0



 



 



5



0



 



 



6



Slight



0



 



 



2



0



 



 



0



 



 



 



 



 



 



 



 



 



Vacuolation, Cortex



 



 



 



 



 



 



 



 



Slight



2



 



 



10



1



 



 



0



 



 



 



 



 



 



 



 



 



Liver



 



 



 



 



 



 



 



 



Number Examined



25



0



0



23



24



2



0



25



  Hypertrophy, Centrilobular



 



 



 



 



 



 



 



 



Minimal



0



 



 



5



1



0



 



7



  Hyperplasia, Bile Duct



 



 



 



 



 



 



 



 



Minimal



1



 



 



5



0



0



 



0



slight



0



 



 



1



0



0



 



0



 



 



 



 



 



 



 



 



 



Kidneys



 



 



 



 



 



 



 



 



Number Examined



25



2



4



23



24



1



1



25



  Accumulation, Hyaline Droplets,



 



 



 



 



 



 



 



 



Slight



3



2



2



12



0



0



0



0



Moderate



0



0



1



7



0



0



0



0



 



 



 



 



 



 



 



 



 



 


: Body weight (g) - group mean values of females before pairing and on GD 0

















































































































Cohort



 



1A + 1B



1A



1B



Bwt, gain



Group



 



Week



Week



Week



GD



during



/Sex



 



10



10



10



0



pairing



Statistics



test



Wi



Wi



Wi



Wi



 



1F



Mean



271



266



275



282



+7



 



 



 



 



 



 



 



2F



Mean



274



275



272



278



+6



 



 



 



 



 



 



 



3F



Mean



278



281



275



278



+3



 



 



 



 



 



 



 



4F



Mean



264



267



261



263**



+2



 



 



 



 



 



 



 



 


 


: Hexylene glycol-Related Effects in Organ Weight Parameters - Terminal Sacrifice (F1)














































































































































































































































 



Hexylene glycol



 



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



250



800



0



100



250



800



Adrenal Glands



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.061



0.061



0.066



0.073**



0.063



0.067



0.072**



0.077**



Body Weight Ratio (%)



0.013



0.014



0.014*



0.016**



0.024



0.025



0.027*



0.030**



Kidneys



 



 



 



 



 



 



 



 



Absolute Weight (g)



3.39



3.48



3.60



3.87**



1.86



1.97



2.00



2.03*



Body Weight Ratio (%)



0.72



0.77**



0.79**



0.86**



0.71



0.73



0.74*



0.78**



Liver



 



 



 



 



 



 



 



 



Absolute Weight (g)



16.6



16.0



17.2



20.1**



9.1



9.9



10.0



11.4**



Body Weight Ratio (%)



3.52



3.54



3.77



4.46**



3.49



3.65



3.69



4.37**



Prostate



 



 



 



 



 



 



 



 



Absolute Weight (g)



1.02



1.09



1.08



1.11



 



 



 



 



Body Weight Ratio (%)



0.22



0.24



0.24



0.25*



 



 



 



 



Seminal Vesicles



 



 



 



 



 



 



 



 



Absolute Weight (g)



1.81



1.91



1.94



1.94



 



 



 



 



Body Weight Ratio (%)



0.39



0.42*



0.42*



0.43*



 



 



 



 



Ovaries



 



 



 



 



 



 



 



 



Absolute Weight (g)



 



 



 



 



0.090



0.103*



0.106*



0.101*



Body Weight Ratio (%)



 



 



 



 



0.034



0.038*



0.039**



0.039**


           

 


 


Incidence and Severity of Hexylene glycol-Related Microscopic Findings - Terminal Sacrifice (F1)
























































































































































































































































































































































Sex



Hexylene glycol



Males



Females



Dose Level (mg/kg/day)



0



100



250



800



0



100



250



800



Adrenal Glands



 



 



 



 



 



 



 



 



Number Examined



20



0



0



20



20



0



0



20



 Hypertrophy, Cortical, Diffuse



 



 



 



 



 



 



 



 



Minimal



0



 



 



10



2



 



 



13



 



 



 



 



 



 



 



 



 



Vacuolation, Cortex



 



 



 



 



 



 



 



 



Slight



2



 



 



9



0



 



 



0



 



 



 



 



 



 



 



 



 



Liver



 



 



 



 



 



 



 



 



Number Examined



20



1



0



20



20



0



0



20



  Hypertrophy, Centrilobular



 



 



 



 



 



 



 



 



Minimal



0



0



 



6



0



 



 



9



  Hyperplasia, Bile Duct



 



 



 



 



 



 



 



 



Minimal



4



0



 



6



0



 



 



0



 



 



 



 



 



 



 



 



 



Kidneys



 



 



 



 



 



 



 



 



Number Examined



20



5



2



20



20



3



2



20



  Accumulation, Hyaline Droplets,



 



 



 



 



 



 



 



 



Slight



1



0



2



9



0



0



0



0



Moderate



0



0



0



6



0



0



0



0



  Basophilia, Tubular, Focal



 



 



 



 



 



 



 



 



Minimal



9



2



0



5



0



1



1



1



Slight



0



0



0



1



0



0



0



0



  Basophilia, Tubular, Multifocal



 



 



 



 



 



 



 



 



Minimal



0



0



1



2



0



0



0



0



Slight



0



0



0



5



0



0



0



0



  Casts, Granular



 



 



 



 



 



 



 



 



Minimal



0



0



0



2



0



0



0



0



Slight



0



0



0



1



0



0



0



0


Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the No Observed Adverse Effect Level (NOAEL) for systemic toxicity which has the potential to be harmful to human health in the F0 parent animals and the F1 Cohort 1A and 1B adult animals is 800 mg/kg/day. The NOAEL for kidney changes in F0 and F1 males consistent with the accumulation of alpha 2µ-globulin which was deemed adverse for the animals (but this finding is not generally considered relevant to human health) is 100 mg/kg/day. The NOAEL for reproductive performance of the F0 and F1B parent animals and survival and growth of the F1 and F2 offspring was concluded to be 250 mg/kg/day, based on transient reduced survival of the F1 and F2 offspring during the early post-natal period
Executive summary:

The purpose of this study was to assess the influence of Hexylene glycol on reproductive performance when administered continuously by oral gavage to Sprague Dawley rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation and estrous cycles and one cohort was assessed for reproductive performance (as requested in ECHA Decision number: TPE-D-2114453322-58-01/F).


In the F0 generation, three groups of 25 male and 25 female rats received Hexylene glycol at dose levels of 100, 250 or 800 mg/kg/day at a volume dose of 5 mL/kg/day. Males were treated for two weeks before pairing, up to necropsy after the litters were weaned. Females were treated for two weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. In the F1 generation, 20 males and 20 females were treated from weaning to scheduled termination in adulthood (relevant to each cohort) at the same dose levels and volume-dose as the F0 generation. A similarly constituted Control group in each generation received the vehicle, purified water, at the same volume dose as the treated groups.


For the F0 generation data were recorded on clinical observations, detailed physical examination and arena observations, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance. Clinical pathology (hematology, blood chemistry and urinalysis) and thyroid-related hormones, sperm assessment, organ weight, macroscopic pathology and microscopic pathology investigations were performed.


For F1 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 of age. Serum samples that were collected from selected offspring on Day 22 of age were analyzed for thyroid-related hormones.


The F1 generation comprised of two cohorts:


For F1 Cohort 1A, data were recorded on clinical condition, detailed physical examination and arena observations, body weight, food consumption, sexual maturation and estrous cycles. Clinical pathology (hematology, blood chemistry and urinalysis) and thyroid-related hormones, sperm assessment, ovarian follicle and corpora lutea counts, organ weight, macroscopic pathology, full microscopic pathology and immunophenotyping investigations were performed.


For F1 Cohort 1B, data was recorded on clinical condition, detailed physical examination and arena observations, body weight, food consumption, sexual maturation, estrous cycles, mating performance, fertility, gestation length and gestation index, organ weight and macroscopic and microscopic pathology investigations were performed.


For F2 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 of age.


Results


There were four unscheduled deaths (one male at 100 mg/kg/day, one male at 250 mg/kg/day and two males at 800 mg/kg/day) in the F0 generation and one unscheduled death (one Control male) in the F1 generation, none were treatment related.


There were no signs attributed to oral gavage administration and no clinical signs (detailed physical examination and arena observations) attributable to treatment in the F0 or F1 adults.


Body weight gain and food consumption were unaffected by treatment during the two (F0) and 10 (F1) week pre-pairing periods and during gestation or lactation and in the F1 and F2 offspring body weights were unaffected by parental treatment.


Pre-pairing estrous cycles in the F0 and F1 females, estrous cycles at 10 weeks of age (F1) and estrous cycles following weaning of litters and before termination (F0 and F1) were unaffected by treatment.


Mating performance and fertility, gestation length and gestation index in the F0 and F1adults were unaffected by the treatment. The numbers of implantations and total offspring born were unaffected by parental treatment at 100, 250 or 800 mg/kg/day.


In the F1 litters, the live birth index was marginally low, reflecting a higher number of litters with offspring deaths up to Day 1 of age at 800 mg/kg/day and this was considered treatment related. Subsequent survival of the offspring was unaffected. In the F2 litters, a higher number of litters had offspring deaths between Days 1 and 4 of age, resulting in a decreased viability index and a marginally low litter size and this was considered treatment related.


The ratio of male to female offspring and the ano-genital distance of the F1 and F2 offspring were considered unaffected by treatment and no F1 male offspring had nipples. One F2 male at 100 mg/kg/day had one nipple; however this was considered not to be toxicologically important.


Absolute body weights of selected F1 males and females at 800 mg/kg/day were slightly but statistically significantly low on Day 1 of the formal F1 generation, when compared with Control.


Sexual maturation of F1 males and females and the period of time to first estrus following maturation was unaffected by treatment.


Hematology, blood chemistry and urinary parameters were not adversely affected by treatment at any dose in the F0 or F1. Adult serum TSH and T4 concentrations were unaffected by treatment in the F0 (adults) or F1 (offspring on Day 22 of age or adults).


Sperm count, motility and motion and morphology; splenic immunophenotyping parameters, ovarian follicle and corpora lutea counts were unaffected by treatment in the F1.


Administration of Hexylene glycol at 800 mg/kg/day resulted in centrilobular hepatocyte hypertrophy, an adaptive change and therefore considered not to be an adverse effect, in the liver of males and females in both F0 and F1 generations and correlated with an increase in organ weight.


Administration of Hexylene glycol resulted in hypertrophy, an adaptive change and therefore considered not to be an adverse effect, of the cortex of the adrenal glands of males and females given 800 mg/kg/day. This correlated with an increase in organ weight and hypertrophy.


Administration of Hexylene glycol to males at 100, 250 or 800 mg/kg in the F0 generation and 250 or 800 mg/kg/day in the F1 generation resulted in cortical vacuolation of the adrenal glands, an adaptive change and therefore considered not to be an adverse effect.


Administration of Hexylene glycol at all dose levels caused an increase in hyaline droplets in the kidney of males. This was considered non-adverse in the F0 generation and in the F1 males given 100 mg/kg/day. Adverse changes were apparent in F1 males given 250 or 800 mg/kg/day (basophilic tubules and granular casts), however these changes are not thought to be significant to man.


Other organ weight changes were considered not to be pathologically significant as there was no associated histopathological change.


Organ weights of the unselected F1 and the F2 offspring on Day 22 of age were unaffected by parental treatment and there were no macroscopic findings related to treatment in the F0 (adults), F1 (offspring and adults), F2 offspring.


Conclusion


Based on the results of this study it is concluded that the No Observed Adverse Effect Level (NOAEL) for systemic toxicity which has the potential to be harmful to human health in the F0 parent animals and the F1 Cohort 1A and 1B adult animals is 800 mg/kg/day. The NOAEL for kidney changes in F0 and F1 males consistent with the accumulation of alpha-2µ-globulin which was deemed adverse for the animals (but this finding is not generally considered relevant to human health) is 100 mg/kg/day. The NOAEL for reproductive performance of the F0 and F1B parent animals and survival and growth of the F1 and F2 offspring was concluded to be 250 mg/kg/day, based on transient reduced survival of the F1 and F2 offspring during the early post-natal period.