Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 November 2016 - 30th April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 15 December 2016 and 30th April 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test item: H-32
Test item identity (including alternative names): Decanoic acid, ester with 2,2-bis(hydroxymethyl) - 1,3-propanediol 2-ethylhexanoate octanoate
CAS number: 68130-25-6
Appearance: Pale yellow liquid
Storage conditions: At ambient temperature (15 to 25°C) in the dark
Batch number: 64296
Expiry date: 30 September 2018
Purity: 99%<
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.

Archive sample: A 6.0 g representative sample was taken from each batch of test item. This sample was placed in a well closed glass
container and stored in the archives under the same conditions as the bulk material.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD) rat.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males 71 days old. Females 85 days old.
- Weight at study initiation: Males 322 to 384g. Females 239 to 291g.
- Fasting period before study: No
- Housing:Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet (e.g. ad libitum):SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. (NON restricted)
- Water (e.g. ad libitum):Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.(NON restricted)
- Acclimation period:Males: 6 days prior to the commencement of treatment. Females: 20 days prior to the commencement of treatment
ENVIRONMENTAL CONDITIONS
- Temperature (°C):Monitored and maintained within the range of 20-24ºC
- Humidity (%):40-70%.
- Air changes:Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 hours light : 12 hours dark.
IN-LIFE DATES: From: 15 February 2017 To: 30 April 2017

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
Propylene glycol.

Method of preparation
Required amounts of test item were weighed into suitable containers. Starting with the lowest concentration, a series of concentrations were obtained by addition of vehicle, with stirring, until approximately 50% of the final volume was obtained. After uniformity of mixture was observed, further vehicle was then added, with stirring, until final volume was obtained.

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.

- Concentration in vehicle: 0, 20, 66 or 200 mg/mL
- Amount of vehicle: 5mL/kg

Details on mating procedure:

- M/F ratio per cage:1:1 from within the same treatment groups.
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis

Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. Eight days stability was confirmed when stored refrigerated (2 to 8°C) or one day when stored at room temperature (15 to 25°C).

Achieved concentration
Samples of each formulation prepared for administration in Weeks 1 and 4 (males) of treatment and on Day 12 of lactation (females) were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

At least 4 weeks

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

330 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The study consisted of one control and three treated groups identified as follows.

The F0 generation of the study was identified as follows:

Group Treatment Dose (mg/kg/day)# Number of animals Animal numbers
Male Female Male Female
1 Control 0 10 10 21-30 61-70
2 H-32 100 10 10 11-20 51-60
3 H-32 330 10 10 31-40 71-80
4 H-32 1000 10 10 1-10 41-50

# Expressed in terms of test material as supplied.

Some serial observations needed to be performed without the knowledge of the treatment group; therefore the animal numbering system was such that it was not easy to identify a treatment group from the animal number.

The F1 generation received no direct administration of the test item, H-32. Any exposure to the test item or metabolites was through the mother to the offspring in utero and/or through the milk.

Administration
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed
directly.

Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Once daily at approximately the same time each day.
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Positive control:

None specified

Parental animals: Observations and examinations:

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 occupants. 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 males Week 1 - daily
Week 2 onwards - once each week
F0 females Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation.
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 and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 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. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”. 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.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were moved into individual cages prior to transport to the testing room. The cage labels on these individual cages showed only the study and animal number. Animals were not necessarily all
tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.
The following measurements, reflexes and responses were recorded:

Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity
During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.

Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

Body Weight
The weight of animals was recorded as follows:

F0 males Weekly during acclimatization.
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter. On the day of necropsy.

F0 females Weekly during acclimatization.
Before dosing on the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

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

F0 animals Weekly, from the day that treatment commenced. 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 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.

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

Mating Procedure
Pairing commenced After a minimum of two weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups.
Duration of pairing Up to two weeks.
Daily checks for evidence Ejected copulation plugs in cage tray and sperm in the vaginal
of mating 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.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears
From beginning of treatment until animals were paired for mating, using cotton swabs.
Wet smears
Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.

After pairing until mating.
For four days before scheduled termination (nominally Days 11 to 14 of lactation).

Parturition Observations and Gestation Length
Duration of gestation Time elapsing between the detection of mating and commencement of parturition.

Parturition observations
From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Litter observations:

Clinical observations
Examined at approximately 24 hours after birth (Day 1 of age)
and then daily thereafter for evidence of ill health or reaction to
maternal treatment; these were on an individual offspring basis
or for the litter as a whole, as appropriate.

Litter size
Daily records were maintained of mortality and consequent
changes in litter size from Days 1-13 of age.

Sex ratio of each litter
Recorded on Days 1, 4, 7 and 13 of age.

Individual offspring body weights
Days 1, 4, 7 and 13 of age.

Ano-genital distance
Day 1 - all F1 offspring.

Nipple/areolae count
Day 13 of age - male offspring.

Postmortem examinations (parental animals):

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Time of Necropsy
F0 males After Week 6 investigations completed.
F0 females failing
to produce a
viable litter Day 25 after mating.

F0 females Day 14 of lactation (following terminal blood sampling).
F1 offspring Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.

Females
The following were recorded:
Each uterine horn Number of implantation sites was counted and confirmed if
none were visible at visual inspection.

Postmortem examinations (offspring):

Offspring
Premature deaths Where possible, a fresh macroscopic examination (external
and internal) with an assessment of stomach for milk content
was performed.

F1 offspring on Day 4 of Blood sampling required (See Section 3.6.11).
age Externally normal offspring discarded without examination.
Externally abnormal offspring examined, and retained pending
possible future examination.


F1 offspring on Day 13 of Blood sampling required (See Section 3.6.11).
age All animals (but not including those selected for thyroid
hormone analysis) were subject to an external macroscopic
examination; particular attention was paid to the external
genitalia. Animals observed with external abnormalities were
retained pending possible future examination
Thyroid glands were preserved from one male and one female
in each litter.
Animals selected for thyroid hormone analysis: externally
normal offspring were discarded without examination.
Externally abnormal offspring were examined.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these
tests, whether significant or non-significant, are presented on the relevant tables. For some
parameters, including estrous cycles before treatment, pre-coital interval and stage of estrous
at termination the similarity of the data was such that analyses were not considered to be
necessary:

All statistical analyses were carried out separately for males and females. For all other adult
parameters, the analyses were carried out using the individual animal as the basic
experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis
for statistical analysis and biological significance was assessed with relevance to the severity
of the anomaly and the incidence of the finding within the background control population.

Clinical signs:

no effects observed

Description (incidence and severity):

Animal 1F 61 was found dead on Day 14 of Lactation. On Day 12 of Lactation dose observations of chin rubbing and salivation were seen. Macropathology findings revealed clear fluid in the stomach, dark areas on the glandular mucosa of the stomach and pale areas on the ventricles of the heart. Microscopic findings in the heart of myocardial mineralisation correlate with the pale areas seen macroscopically. Cortical tubular degeneration and mineralisation were seen in the kidneys with mucosal degeneration and mineralisation also seen in the stomach. Dark areas seen in the stomach macroscopically correlated with the mucosal erosion seen microscopically. Involution atrophy was seen in the thymus and neuronal vacuolation in the brain. Factor contributing to death was the mineralisation and degeneration seen.

No clinical signs or dose observations related to treatment of H-32 were seen in the treatment, gestation or lactation periods.

No detailed physical examination and arena observations related to treatment of H-32 were seen in the treatment, gestation or lactation periods.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Animal 1F 61 was found dead on Day 14 of Lactation. On Day 12 of Lactation dose observations of chin rubbing and salivation were seen. Macropathology findings revealed clear fluid in the stomach, dark areas on the glandular mucosa of the stomach and pale areas on the ventricles of the heart. Microscopic findings in the heart of myocardial mineralisation correlate with the pale areas seen macroscopically. Cortical tubular degeneration and mineralisation were seen in the kidneys with mucosal degeneration and mineralisation also seen in the stomach. Dark areas seen in the stomach macroscopically correlated with the mucosal erosion seen microscopically. Involution atrophy was seen in the thymus and neuronal vacuolation in the brain. Factor contributing to death was the mineralisation and degeneration seen.

No clinical signs or dose observations related to treatment of H-32 were seen in the treatment, gestation or lactation periods.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment of H-32 has had no effect on bodyweight measurements in the treatment or gestation period. It was noted that after the pairing period Cage No.3 containing male animals 11-15 (Group 2 (100 mg/kg/day)) were behaving aggressively to each other once returned to their home cage. It is thought that consequently the body weight loss seen for animals 11, 13 and 14 from Week 2 to Week 4 of study is due to behavior seen after the temporary separation period.

During the lactation period all groups of treated females had slightly lower than control body weights, however there was no dose relationship and bodyweight gain was similar to controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Treatment of H-32 had no effect on food consumption measurements in the treatment, gestation or lactation periods.

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):

Haematology investigations after 5 weeks of treatment revealed slightly but statistically lower than control mean cell volume in all groups of treated males, however no dose relationship is present. Females receiving 1000 mg/kg/day had a marginally but statistically lower than control red blood cell count.

All other haematological differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry investigations after 5 weeks of treatment revealed statistically lower than control creatinine, calcium and albumin in male animals receiving 100 mg/kg/day, however no dose relationship was apparent.

All other biochemical differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The microscopic examination performed after 5 weeks of treatment revealed no test item related lesions.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The microscopic examination performed after 5 weeks of treatment revealed no test item related lesions.

Incidental Findings
Mineralisation sometimes accompanied by degeneration was seen in the heart, kidneys and stomach of females across the groups at a similar level in control and treated animals. This is considered to be a background lesion based on recent background data and is now considered unrelated to test-item or vehicle.
The incidence and distribution of all other findings were considered to be unrelated to treatment.

Other effects:

not specified

Description (incidence and severity):

The sensory reactivity observations conducted during Week 5 of treatment revealed no findings which were considered treatment related.

The motor activity assessment conducted during Week 5 of treatment revealed no treatment related effects in all animals at all dose levels.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

All females allocated to the study showed regular 4 or 5 day estrus cycles prior to the start of treatment.
During treatment one control female and one female receiving 1000 mg/kg/day showed an irregular cycle, the remaining animals were regular.

At termination, all reproductive phase females showed diestrous.

There was no effect of treatment on the pre coital interval: all animals mated within four days of pairing at the first estrous.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance and fertility was 80% for control animals, all treated groups were 100%.
There was no effect of treatment on gestation length or the gestation index.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed among the F1 litters that were clearly attributable to parental treatment with H-32.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was considered to be no effect of treatment on litter size, offspring survival or the sex ratio.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of treatment on mean body weights of male and female offspring in the 100 or 330 mg/kg/day groups on Day 1 of age, or on subsequent bodyweight gain until termination on Day 13 of age.

Mean bodyweight of male and female offspring in the 1000 mg/kg/day group on Day 1 of age was comparable to Control offspring. However the overall body weight change from Days 1 to 13 for both sexes was marginally lower (1-3%) than 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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings observed in the offspring that died prior to scheduled termination or among those offspring killed on Day 13 of age that were attributable to parental treatment with H-32.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Ano-genital distance in male and female offspring was unaffected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Key result

Reproductive effects observed:

no

Conclusions:

It was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity and reproductive/developmental toxicity was 1000 mg/kg/day. Administration of H-32 at dose levels of 100, 330 or 1000 mg/kg/day had no effect on clinical condition, body weight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, mating performance and fertility and gestation length, histopathology, litter size, offspring survival, sex ratio, offspring growth, offspring clinical signs, ano-genital distances or macropathology. All other differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Executive summary:

The purpose of this study was the assessment of general systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of H-32 by oral administration for at least four weeks.

Three groups of ten male and ten female Sprague Dawley rats received H-32 at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of six consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the

vehicle, propylene glycol, at the same volume dose as the treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Thyroid hormone levels were analysed for Day 13 offspring.

Results

Analyses of samples for thyroxine (T4) obtained from Main study male animals and F1 offspring on Day 13 of age did not reveal any differences that could be attributed to treatment; therefore further assessment of T4 and thyroid stimulating hormone (TSH) was

considered unnecessary.

There were no deaths related to treatment with H-32. Administration of H-32 at dose levels of 100, 330 or 1000 mg/kg/day had no effect on clinical condition, body weight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, mating performance and fertility and gestation length or the

microscopic appearance of a full range of tissues/organs.

All females allocated to the study showed regular 4 or 5 day estrus cycles prior to the start of treatment. During treatment one control female and one female receiving 1000 mg/kg/day showed an irregular cycle, the remaining animals were regular. At termination, all reproductive phase females showed diestrous.

At haematology evaluation all groups of treated males had slightly but statistically lower than control mean cell volume, however no dose relationship was present. Females receiving 1000 mg/kg/day had a marginally but statistically lower than control red blood cell count. At blood chemistry evaluation study males receiving 100 mg/kg/day had statistically lower than control creatinine, calcium and albumin, however no dose relationship was apparent. There were no differences in organ weights which were considered to be related to treatment. Macroscopic findings included dark areas in the glandular mucosa of the stomach in one control female and several of the females in each treated group. Hair loss was seen in females treated at 1000 mg/kg/day, one female treated at 100 mg/kg/day and one control

female.

There was no effect of H-32 on litter size, offspring survival, sex ratio, offspring clinical signs, ano-genital distances or macropathology.

Mean bodyweight of male and female offspring in the 1000 mg/kg/day group on Day 1 of age was comparable to Control offspring. However the overall body weight change from Days 1 to 13 for both sexes was marginally lower (3% lower) than Control. There was no effect of treatment on mean body weights of male and female offspring in the 100 or 330 mg/kg/day groups on Day 1 of age, or on subsequent bodyweight gain until termination on Day 13 of age.

Conclusion

It was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity and reproductive/developmental toxicity was 1000 mg/kg/day. Administration of H-32 at dose levels of 100, 330 or 1000 mg/kg/day had no effect on clinical condition, body weight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, mating performance and fertility and gestation length, histopathology, litter size, offspring survival, sex ratio, offspring clinical signs, ano-genital distances or macropathology. All other differences from controls observed during the treatment period were minor or lacked

dose relationship and were therefore attributed to normal biological variation.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Version adopted 29 July 2016

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

EC Number:

268-582-0

EC Name:

Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

Cas Number:

68130-25-6

Molecular formula:

C10 H20 O2 . x C8 H16 O2 . x C8 H16 O2 . x C5 H12 O4

IUPAC Name:

Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

Test material form:

liquid

Details on test material:

Identification: H-32
CAS No.: 68130-25-6
Batch: 64296
Purity: > 99%
Appearance: Pale yellow liquid
Expiry Date: 30 September 2018
Storage Conditions: At room temperature
Stability in Solvent: Not relevant
Purpose of Use: Industrial chemical

Specific details on test material used for the study:

Test item: H-32
Test item identity (including alternative names): Decanoic acid, ester with 2,2-bis(hydroxymethyl) - 1,3-propanediol 2-ethylhexanoate octanoate
CAS number: 68130-25-6
Appearance: Pale yellow liquid
Storage conditions: At ambient temperature (15 to 25°C) in the dark
Batch number: 64296
Expiry date: 30 September 2018
Purity: 99%<
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.

Archive sample: A 6.0 g representative sample was taken from each batch of test item. This sample was placed in a well closed glass
container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males: 71 days; Females: 85 days
- Weight at study initiation: Males: 322 to 384g; Females: 239 to 291g
- Fasting period before study: No fasting prior to study intitiation
- Housing:Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet: Access non-restricted (removed overnight before blood sampling for hematology and blood chemistry investigations).
- Water: Access non-restricted
- Acclimation period:Males: 6 days prior to the commencement of treatment. Females: 20 days prior to the commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: 12 hours light: 12 hours dark

IN-LIFE DATES: From: 07 March 2017 To: 30 April 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

Required amounts of test item were weighed into suitable containers. Starting with the lowest concentration, a series of concentrations were obtained by addition of vehicle, with stirring, until approximately 50% of the final volume was obtained. After uniformity of mixture was observed, further vehicle was then added, with stirring, until final volume was obtained.

VEHICLE
- Concentration in vehicle: 0, 20, 66, or 200mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males: At least five weeks (2 weeks before pairing, up to two week during the mating period, one week of observations after the end of the mating period)
Females: Two weeks before mating, up to 2 weeks' mating period then up to 25 days post-mating (females failing to provide a viable litter) or day 14 of lactation.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A preliminary 14-day repeat dose toxicity study was conducted (Envigo study number XF08VY) prior to study initiation. This preliminary study concluded that doses up to 1000 mg/kg/day were well tolerated.

- Rationale for animal assignment (if not random):
Aoolcation on arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced by Study Management to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
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 occupants. 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.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males Weekly during acclimatization.
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.

F0 females Weekly during acclimatization.
Before dosing on the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

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

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes - isoflurane
- Animals fasted: Yes (overnight withdrawal of food)
- How many animals: five per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: Yes (overnight withdrawal of food)
- How many animals: five per group
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes

Detailed physical examination and arena observations
Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. 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.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation.
The following measurements, reflexes and responses were recorded: Approach response, pinna reflex, auditory startle reflex, tail pinch response, grip response.

Motor activity
During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Refer to tables 3 and 4 for details of tissues examined

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the control group was found dead on day 14 of lactation

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment of H-32 has had no effect on bodyweight measurements in the treatment or gestation period. It was noted that after the pairing period a cage containing male animals 11-15 (Group 2 (100 mg/kg/day)) were behaving aggressively to each other once returned to their home cage. It is thought that consequently the body weight loss seen for animals 11, 13 and 14 from Week 2 to Week 4 of study is due to behavior seen after the temporary separation period.

During the lactation period all groups of treated females had slightly lower than control body weights, however there was no dose relationship and bodyweight gain was similar to controls.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology investigations after 5 weeks of treatment revealed slightly but statistically lower than control mean cell volume in all groups of treated males, however no dose relationship is present. Females receiving 1000 mg/kg/day had a marginally but statistically lower than control red blood cell count.

All other haematological differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry investigations after 5 weeks of treatment revealed statistically lower than control creatinine, calcium and albumin in male animals receiving 100 mg/kg/day, however no dose relationship was apparent.

All other biochemical differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No detailed physical examination and arena observations related to treatment of H-32 were seen in the treatment, gestation or lactation periods.

The sensory reactivity observations conducted during Week 5 of treatment revealed no findings which were considered treatment related.

The motor activity assessment conducted during Week 5 of treatment revealed no treatment related effects in all animals at all dose levels.
It was noted that after the pairing period Cage No.3 containing male animals 11-15 (Group 2 (100 mg/kg/day)) were behaving aggressively to each other once returned to their home cage. It is thought that the statistical significant low levels of activity seen throughout the 1-hour recording period for these males was due to the signs of aggression seen prior to activity testing, this would suggest the animals were quite stressed and/or tired. These findings however are considered to be unrelated to treatment of H-32.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute and body weight adjusted kidney and thymus weights were higher than control in male animals receiving 330 or 1000 mg/kg/day, however the difference did not attain statistical significance. Liver weights were marginally higher for male animals receiving 330 or 1000 mg/kg/day; however no dose relationship was apparent.

Absolute and body weight adjusted adrenal weights were marginally higher than control in lactating females animals receiving 330 or 1000 mg/kg/day, however no statistical significance was obtained.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic examination performed after 5 weeks of treatment revealed the following changes in the stomach and hair loss.

Stomach
Dark areas were seen in the glandular mucosa of treated females and in one control female.
Refer to table 5 for details.

Skin
Hair loss was seen in females treated at 1000 mg/kg/day, one female treated at 100 mg/kg/day and one control female.
Refer to table 6 for details.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Incidental Findings
Mineralisation sometimes accompanied by degeneration was seen in the heart, kidneys and stomach of females across the groups at a similar level in control and treated animals. This is considered to be a background lesion based on recent background data and is now considered unrelated to test-item or vehicle.
The incidence and distribution of all other findings were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 5: Summary of findings in the stomach for animals killed after at least 5 weeks of treatment

Group / Sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	330	1000	0	100	330	1000
Dark area(s)	0	0	0	0	1	3	4	3
Number of tissues examined	10	10	10	10	7	10	10	10

 

Table 6: Summary of findings in the skin for animals killed after at least 5 weeks of treatment

Group / Sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	330	1000	0	100	330	1000
Hair loss	0	0	0	0	1	1	0	4
Number of tissues examined	10	10	10	10	7	10	10	10

Applicant's summary and conclusion

Conclusions:

It was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity and reproductive/developmental toxicity was 1000 mg/kg/day. Administration of H-32 at dose levels of 100, 330 or 1000 mg/kg/day had no effect on clinical condition, body weight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, mating performance and fertility and gestation length, histopathology, litter size, offspring survival, sex ratio, offspring growth, offspring clinical signs, ano-genital distances or macropathology. All other differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Executive summary:

The purpose of this study was the assessment of general systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of H-32 by oral administration for at least four weeks.

Three groups of ten male and ten female Sprague Dawley rats received H-32 at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of six consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the

vehicle, propylene glycol, at the same volume dose as the treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Thyroid hormone levels were analysed for Day 13 offspring.

Results

Analyses of samples for thyroxine (T4) obtained from Main study male animals and F1 offspring on Day 13 of age did not reveal any differences that could be attributed to treatment; therefore further assessment of T4 and thyroid stimulating hormone (TSH) was

considered unnecessary.

There were no deaths related to treatment with H-32. Administration of H-32 at dose levels of 100, 330 or 1000 mg/kg/day had no effect on clinical condition, body weight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, mating performance and fertility and gestation length or the

microscopic appearance of a full range of tissues/organs.

All females allocated to the study showed regular 4 or 5 day estrus cycles prior to the start of treatment. During treatment one control female and one female receiving 1000 mg/kg/day showed an irregular cycle, the remaining animals were regular. At termination, all reproductive phase females showed diestrous.

At haematology evaluation all groups of treated males had slightly but statistically lower than control mean cell volume, however no dose relationship was present. Females receiving 1000 mg/kg/day had a marginally but statistically lower than control red blood cell count. At blood chemistry evaluation study males receiving 100 mg/kg/day had statistically lower than control creatinine, calcium and albumin, however no dose relationship was apparent. There were no differences in organ weights which were considered to be related to treatment. Macroscopic findings included dark areas in the glandular mucosa of the stomach in one control female and several of the females in each treated group. Hair loss was seen in females treated at 1000 mg/kg/day, one female treated at 100 mg/kg/day and one control

female.

There was no effect of H-32 on litter size, offspring survival, sex ratio, offspring clinical signs, ano-genital distances or macropathology.

Mean bodyweight of male and female offspring in the 1000 mg/kg/day group on Day 1 of age was comparable to Control offspring. However the overall body weight change from Days 1 to 13 for both sexes was marginally lower (3% lower) than Control. There was no effect of treatment on mean body weights of male and female offspring in the 100 or 330 mg/kg/day groups on Day 1 of age, or on subsequent bodyweight gain until termination on Day 13 of age.

Conclusion

It was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity and reproductive/developmental toxicity was 1000 mg/kg/day. Administration of H-32 at dose levels of 100, 330 or 1000 mg/kg/day had no effect on clinical condition, body weight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, mating performance and fertility and gestation length, histopathology, litter size, offspring survival, sex ratio, offspring clinical signs, ano-genital distances or macropathology. All other differences from controls observed during the treatment period were minor or lacked

dose relationship and were therefore attributed to normal biological variation.