2-Propenoic acid, 2-methyl-, tetracosyl ester, branched

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:

06 December 2018 - 27 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

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)

Deviations:

yes

Remarks:

These deviations were considered not to have compromised the validity or integrity of the study.

Principles of method if other than guideline:

The following deviations from the agreed study plan occurred: Body weight: at the beginning of the treatment period, the body weights of females were between 251 g and 326 g, instead of between 220g and 320 g. Female P22813 (group 4) was not weighed before its premature euthanasia. On several occasions, clinical observations of animals were performed outside the authorized time of 1 hour (± 15 minutes) after dosing (range of -8 to +7 minutes). On Day 14 p.p., blood for haematology was clotted for females P22791 (group 2), P22797 (group 3), P22807 and P22817 (group 4), but samples were not collected from the remaining females in each group as stated in the study plan, by error. These deviations were considered not to have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, 2-methyl-, tetracosyl ester, branched

EC Number:

292-146-9

EC Name:

2-Propenoic acid, 2-methyl-, tetracosyl ester, branched

Cas Number:

90552-24-2

Molecular formula:

C28H54O2

IUPAC Name:

2-Propenoic acid, 2-methyl-, tetracosyl ester(branched)

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected as background data from previous studies are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) Males 10 wks; Females 11 wks
- Weight at study initiation: (P) Males: 432g to 477g, Females: 251 to 326g
- Housing: Animals were individually housed, except during mating (male + female) and lactation (female + pups), in polycarbonate cages (Tecniplast 2154, 48 x 26.5 x 21 cm, 940 cm2) with stainless steel lids and containing autoclaved sawdust. Toward the end of gestation and during lactation, females and their litters were provided with autoclaved wood shavings as nesting material. Each cage contained a rat hut and a piece of wood for the environmental enrichment of the animals. Cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): ad libitum access to SSNIFF rat/mouse pelleted maintenance diet, batch Nos. 14535074 and 94340868 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): ad libitum access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: males 8 days before treatment, females 6 days before the beginning of estrous cycle monitoring during the pre-treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approx. 8 to 15 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time.

Vehicle:

peanut oil

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle: The vehicle for preparation of test item dose formulations was selected based on previous experimental work and/or preliminary studies.
- Concentration in vehicle: Solution from 3 to 300 mg/mL in the vehicle. Measured concentration = nominal concentration ± 10%.
- Amount of vehicle: A constant dosage volume of 5 mL/kg/day was used.
- Lot/batch no.: MKBX4739V

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical technique: Gas Chromatography with Flame Ionization detection (GC-FID). The actual test item concentrations in the dose formulations were determined in Weeks 1, 3 and 6 using the validated GC-FID method. Acceptance criterion: Measured concentration = nominal concentration ± 10%. The actual test item concentrations in the analyzed dose formulations (i.e. in Weeks 1, 3 and 6) remained within an acceptable range of variation (-2.4% to +2.6%) when compared with the nominal values (nominal concentration ± 10% required).

Duration of treatment / exposure:

The dose formulations were administered daily according to the following schedule: Males: 2 weeks before mating, during the mating period (2 weeks), until euthanasia (at least 4 weeks in total). Females were treated for an overall period of 8 to 9 weeks:
2 weeks before mating, throughout mating (up to 2 weeks) and gestation (3 weeks), during lactation until Day 13 p.p. inclusive, until euthanasia for females with no delivery.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a previous study performed in the same species, wherein the test item was administered daily by gavage to five males and five females at dose levels of 0, 100, 300 or 1000 mg/kg/day for 2 weeks. 1000 mg/kg/day was selected as the high-dose level for the present study. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 100 and 300 mg/kg/day).
- Rationale for animal assignment: animals (40 males and 40 females) were selected according to body weight and clinical condition, and allocated to groups (by sex) according to a stratified procedure based on body weight so that the average body weight of each group was similar.
- Fasting period before blood sampling for clinical biochemistry: Overnight (minimum 14 hours)
- Other: animals were identified by an individual ear tattoo. At the beginning of the study, each animal received a unique identity number.

Positive control:

N/A

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day before the treatment period and at least twice a day during the treatment period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Performed on all animals once before the beginning of the treatment period and then once a week until the end of the study.
-Parameters were examined: included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also evaluated.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each male recorded on the first day of treatment (Day 1), then once a week until euthanasia. Body weight of each female recorded on the first day of treatment (Day 1), then once a week until mated.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of euthanasia
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: first five males and lactating females from each group
- Following parameters were examined: Erythrocytes (RBC), Mean cell volume (MCV), Packed cell volume (PCV), Hemoglobin (HB), Mean cell hemoglobin concentration (MCHC), Mean cell hemoglobin (MCH), Thrombocytes (PLT), Leucocytes (WBC), Differential white cell count with cell morphology (neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L),large unstained cells (LUC),monocytes (M)),Reticulocytes (RTC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of euthanasia
- Animals fasted: Yes
- How many animals: First five males and lactating females from each group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Animals that were euthanized as scheduled were evaluated with an FOB once at the end of the treatment period. For females, this was performed on Day 13 p.p.
- Dose groups that were examined: In each group, the first five males and lactating females
- Battery of functions tested: motor activity / other: reactivity to manipulation and to different stimuli

IMMUNOLOGY: No

OTHER:
Thyroid hormones:
- Time schedule for collection of blood: morning between 7.5 and 10 am
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: 14 hours
- How many animals: all animals

Pathology:
- Time schedule for collection of blood: completion of the treatment period: males: after the end of the mating period, females: on Day 14 p.p.
- Animals fasted: 14 hours
- How many animals: all animals

Morbidity and mortality:
-Time schedule for examinations: once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

Medical care:
-Time schedule for examinations: Throughout the study, group 4 female No. P22812 was given medical treatment due to local cutaneous lesions

General clinical observations:
-Time schedule for examinations: From arrival, each animal was observed once a day as part of the routine examinations. From the start of the treatment period, each animal was observed at least once a day, at approximately 1 hour (± 15 minutes) after dosing, for the recording of clinical signs.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

Body weight, food consumption and reproductive data were compared by one-way analysis of variances and Dunnett test or by Fisher’s exact probability test. For Hematology, blood biochemistry, hormones, motor activity, CITOX software was used to perform the statistical analysis of these data. PATHDATA software was used to perform the statistical analysis of organ weight data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Ptyalism observed on a few occasions, in one isolated male at 300 and 1000 mg/kg/day, in one isolated female at 300 mg/kg/day (during the gestation period) and in one isolated female at 1000 mg/kg/day (during the lactation period). This sign is commonly noted when a test item is administered by gavage. All the other clinical signs (i.e. areas of hair loss, cutaneous lesions, scabs, chromodacryorrhea and/or short/broken teeth) were considered to be unrelated to the test item as they were present in control animals, were not dose-related, were reported sporadically in only a few animals or are commonly observed findings in this species and strain.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no unscheduled deaths in males or control females. In females, there were the following unscheduled deaths: Gestation period: At 100 mg/kg/day: Female P22785 was euthanized on Day 19 p.c. for humane reasons as a detectable mass was observed in the urogenital region which would have impeded delivery. This mass was a mammary adenocarcinoma, a type of tumor occasionally observed in females of this age, and displayed no relationship to test item administration. Female P22786 was euthanized on Day 26 p.c. for absence of delivery. No extraordinary clinical signs were observed prior to euthanasia. This absence of delivery was considered spontaneous and unrelated to test item administration. At 300 mg/kg/day Female P22806 was euthanized on Day 24 p.c. due to delivery difficulties. Signs of poor clinical condition i.e. pallor of extremities, piloerection and round back) were observed prior to euthanasia. Lactation Period: At 1000 mg/kg/day Female P22813 was euthanized on Day 2 p.p. as her litter was found dead (10 pups at birth).No remarkable clinical signs were observed prior to euthanasia.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related differences in mean body weight or mean body weight change at any dose level in males or females during the premating, gestation and lactation periods at any dose level. When compared with controls, a slight mean body weight loss was recorded in females given 1000 mg/kg/day at the beginning of the lactation period (-3 g vs. +2 g). This was followed by a higher mean body weight gain during the second recording interval (+18 g vs. +12 g). This difference was not statistically significant, did not impact the mean body weight or the mean body weight gain over the whole lactation period. Therefore these findings were considered to be non adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects on mean food consumption in males or females. Differences between control and test item-treated animals consisted of slightly higher mean food consumption during: the mating period between Days 1 and 8 in females given 100, 300 or 1000 mg/kg/day (+28%, +17% and +17%, respectively) and between Days 8 and 15 in females at 100 and 1000 mg/kg/day (+33% and +39%, respectively), the whole gestation period in females given 100 (up to +19%; p< 0.001 between Days 7 and 14 p.c.) or 1000 mg/kg/day (up to +33%; up to p<0.01) and in females given 300 mg/kg/day (between Days 7 and 14 p.c.: +8%, p<0.05),the lactation period between Days 1 and 8 p.p. in females given 100 mg/kg/day (+12% to +18%) and between Days 1 and 4 p.p. in females given 300 mg/kg/day (+18%). A relationship with the test item was considered to be unlikely as these variations were poorly dose-related, due to isolated individual values (i.e. group 2 females P22784 and P22790 during the premating period, group 4 female P22810 for which spillage was observed throughout the first week of the premating period and group 4 females P22812 and P22813 during the gestation period) and/or there were no correlation with mean body weight change.

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):

There were no test item-related effects on haematology parameters at any dose level. All the differences from controls, namely lower mean platelet count in males and/or females at all dose levels, lower mean neutrophil and/or basophil counts in males and/or females at all dose levels, lower mean lymphocyte count in females at all dose levels, lower mean large unstained cell count in females given 100 or 300 mg/kg/day, lower or higher mean monocyte count in males or females given 1000 mg/kg/day and/or higher mean eosinophil count in males given 100 mg/kg/day, were of low magnitude, of opposite trends, due to the presence of platelet aggregates (artefacts at 100 mg/kg/day), unrelated to microscopic findings and/or poorly dose-related. Therefore, these differences were considered to be unrelated to the test item treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day and when compared with controls, statistically significant, dose-related, higher mean chloride level was noted in males (+1%). In the absence of similar finding in the opposite sex and any correlation with microscopic observations and/or in view of the low magnitude, this difference was considered to be of no toxicological importance. The other differences from controls consisted of low mean total bilirubin and/or low mean biliary acid levels in males and/or females at all dose levels, low mean cholesterol levels in females given 100 or 1000 mg/kg/day and/or high mean triglyceride level in females given 300 mg/kg/day. In view of the low magnitude and the directions of the changes, the absence of related microscopic findings and/or as mean values were not dose-related, these findings were not considered to be toxicologically important.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor Activity: There were no relevant differences between the test item-treated and control groups in motor activity (horizontal movements and rearing) in males or females. Differences from controls were noted in the mean number of horizontal and rearing movements at 100 mg/kg/day (males: +18% and females: -30%, and females: -32%, respectively), at 300 mg/kg/day (males: -15% and females: -28%, and females: -26%, respectively) and in the mean number of horizontal movements at 1000 mg/kg/day (males: +13%). These values were considered to be of no toxicological importance as they were of opposite trends, not statistically significant, poorly dose-related and/or as values remained close to or within the standard deviation of control values. Functional Observation battery: An absence of grooming was recorded in 5/5 females at 100 mg/kg/day (versus 4/5 in control females) and in 5/5 males at 1000 mg/kg/day (versus 3/5 in control males). An absence of urination was noted in 5/5 females at 1000 mg/kg/day (versus 3/5 in control females). Higher mean landing foot splay was recorded in males given 300 or 1000 mg/kg/day (+16% and +18% vs. controls, respectively) whereas lower mean values were recorded in females given 100 or 300 mg/kg/day (-16% and -11% vs. controls, respectively). These findings were considered to be of no toxicological importance as they were only observed in one sex, were poorly dose-related and/or did not correlate with any other findings.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Mild increases in adrenal weights and decreases in ovary weights were recorded in high dose females (both ca. 20%). These differences did not reach statistical significance and had no microscopic correlation and therefore considered spurious.
All other differences amid control and treated groups were unrelated to dose, did not reach statistical significance and did not have histological correlates. They were considered to be the consequence of individual variations and unrelated to the test item.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Unscheduled deaths: High-dose female No. P22813 (1000 mg/kg/day) was euthanized on day 41 (Day 2 pp) as her litter was found dead. The death of the litter was found to be un-related to the test item administration. Mid dose female No. P22806 (300 mg/kg/day) was euthanized on Day 42 because of difficulties to deliver. Low-dose female No. P22785 (100 mg/kg/day) was euthanized for human reasons on Day 36 because of the presence of a 2.5 cm in diameter mass in the urogenital area. This mass was a mammary adenocarcinoma, a type of tumor occasionally observed in females of this age, and bore no relationship to test item administration. Low-dose female No. P22786 (100 mg/kg/day) was euthanized on Day 42 because of absence of delivery. This absence of delivery was considered fortuitous and unrelated to test item administration. At the end of the treatment period all macroscopic observations were spontaneous in nature.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All observations belonged to the spectrum of spontaneous changes in rats of this age and strain and bore no relationship to test item administration.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

At 100 mg/kg/day Female P22785 was euthanized on Day 19 p.c. for humane reasons as a palpable mass was observed in the urogenital region that would have impeded delivery. This mass was a mammary adenocarcinoma, which is a type of tumor, occasionally observed in females of this age, and bore no relationship to test item administration.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The thyroid hormone levels were considered to be unaffected by the test item in males. When compared with controls, there was a slightly higher mean T4 concentration at 1000 mg/kg/day. Higher mean TSH concentrations were also noted in all test item-treated groups. As these differences did not correlate with each other, were of minor magnitude, not statistically significant, poorly dose-related, and/or due to isolated values and/or as individual vales remained within the range of control values, they were considered to be of no toxicological importance.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

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: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

2-Propenoic acid, 2-methyl-, tetracosyl ester (branched), was administered daily by oral gavage to male and female Sprague-Dawley rats for 2 weeks before mating, during mating and for females, throughout gestation and until Day 13 p.p. at the dose levels of 100, 300 or 1000 mg/kg/day. Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 1000 mg/kg/day (high-dose level) in the absence of treatment-related adverse findings.

Executive summary:

A GLP compliant study assessing the potential toxic effects of 2- Propenoic acid, 2-methyl-, tetracosyl ester (branched) henceforth referred to as “test item” was conducted using the OECD guideline 422: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test method. The test item was administered to three groups of 10 male and 10 female Sprague-Dawley rats, daily, by the oral route (gavage) at dose levels of 100, 300 or 1,000 mg/kg bw/day, using a constant dosing volume of 5ml/kg/day. Males were treated for an overall period of at least 4 weeks (2 weeks before mating, during the mating period and until the day before euthanasia). Females were treated for an overall period of 8 to 9 weeks (2 weeks before mating, throughout mating (up to 2 weeks) and gestation (3 weeks) until day 13 post-partum (p.p.) inclusive). A control group consisting of 10 male and 10 females received the vehicle (peanut oil) alone under the same experimental conditions. Concentrations of the test item in the dose formulations were determined using a validated Gas chromatography with flame ionisation detection analytical method in weeks 1, 3 and 6. Animals were observed daily during the treatment period for mortality, morbidity and clinical signs. No test item-related unscheduled deaths were noted. In relation to clinical signs, ptyalism occurred in one isolated male at 300 and 1,000 mg/kg bw/day and in one isolated female at 300 and 1,000 mg/kg bw/day during the gestation or the lactation period, respectively. This sign was not considered as an adverse effect as it is frequently detected when a test item is administered by gavage. Comprehensive clinical observations were conducted on a weekly basis. Body weights and food consumption were recorded weekly during the premating, mating, gestation and lactation periods (food consumption not recorded during mating period). No test-item related effects on mean body weight or body weight change at any dose level were noted. A Functional Observation Battery (FOB) was performed on five animals per sex and group at the end of the treatment period where no test item-related effects on FOB or motor activity data at any dose level were noted. Laboratory investigations (haematology and blood biochemistry) were carried out on designated males and females from each group at the end of the study. No test item-related effects on haematology parameters or blood chemistry at any dose level were noted. Plasma thyroid hormone levels (TSH and T4) were determined at termination in all males. No test item-related effects on T4 or TSH levels in males. The males were euthanized after completion of the mating period (i.e. after 30 days of treatment) and females were euthanized on Day 14 p.p. A full macroscopic post-mortem examination was performed on animals, with particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from five males and lactating females in the control and high-dose groups, in one group 3 female and one group 4 female that were prematurely euthanized and on all macroscopic lesions (all groups). The test item was not associated with any organ weights, macroscopic or microscopic changes. Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was found to be 1,000 mg/kg bw/day (high-dose level) in the absence of treatment-related adverse findings.