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

Endpoint summary

Description of key information

NOAEL (Oral, Rat, Sub-acute) = 1000mg/kg/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

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

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)

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

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

Dose / conc.:

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

Remarks:

Vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 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.

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

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

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

Key result

Critical effects observed:

no

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Available study is a repeat dose toxicity screening study performed according to current OECD test guidelines (OECD422, 2016 revision) in compliance with GLP. Data is considered satisfactory to meet REACH Registration requirements.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a repeat dose toxicity screening study (OECD test guideline 422) conducted in rats, no adverse treatment-related effects were observed at levels up to 1000mg/kg/day. In the absence of identifiable adverse effects no target organ toxicity is indicated and there is no basis for classification for Specific Target Organ Toxicity (STOT-RE).