Hexadecyl palmitate

Endpoint summary

Description of key information

Oral (OECD 422, read across): NOAEL m/f rat ≥ 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Jun - 04 Aug 2013

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)

Version / remarks:

adopted in 22 Mar 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 191 to 204 g (males) and 172 to 179 g (females)
- Housing: From arrival to pairing: animals were housed 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating: animals were housed one male to one female in clear polysulphone cages measuring approximately 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating; the females were transferred to individual solid bottomed cages (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy) for the gestation period and parturition.
- Diet: laboratory rodent diet, 4 RF 21 (Mucedola S.r.l., Settimo Milanese (MI), Italy), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous carboxymethylcellulose (0.5% CMC)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in the vehicle. Formulations were prepared daily.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL for dose levels of 100, 300 and 1000 mg/kg bw/day, respectively
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test substance in the vehicle were verified by gas chromatopgraphy with a flame ionisation detection (FID). Concentration verification was conducted on a weekly basis.

Duration of treatment / exposure:

Males: The daily administration of the test item was started two weeks before mating and lasted until test day 28 to 29, which was one day before sacrifice.
Females: The daily administration of the test item was started two weeks before mating and continued until day 3 post-partum.
Maximum: 54 days of treatment.

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

300 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: based on a 14-day range-finding study (Rossiello, 2013. RTC Study No.: 93730EXT)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily during the study, each animal was observed and any clinical signs recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypes or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).

BODY WEIGHT: Yes
- Time schedule for examinations: females: weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: the weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: daily by visual appraisal

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment
- Anaesthetic used for blood collection: Yes with isofluorane
- Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters examined: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count and differential leucocyte count

Coagulation tests: Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment
- Animals fasted: Yes
- How many animals :5/sex/dose
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, total bilirubin, total cholesterol, creatinine, glucose, urea, total protein, calcium, chloride, potassium, sodium, alanine aminotransferase (ALAT), alkaline phosphatase (AP), aspartate aminotransferase (ASAT), gamma-glutamyltransferase, triglycerides, inorganic phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the study, towards the end of treatment, 5 males and 5 females were randomly
selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli), for assessment of grip strength and for the motor activity was measured (for approximately 5 min).
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad), as well as the assessment of grip strength (Meyer) and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
-Organ weights: adrenal glands, brain (cerebrum, cerebellum, medulla/pons), epididymides, heart, kidneys, liver, ovaries with oviducts, parathyroid glands, prostate gland, seminal vesicles with coagulating glands, spleen, testes, thymus (where present), thyroid and uterus-cervix,
-Fixation: adrenal glands, bone marrow (from sternum), brain (cerebrum, cerebellum, medulla/pons), caecum, clitoral gland, colon, duodenum, epididymides, heart, ileum (including Peyer’s patches), jejunum, kidneys, liver, lungs (including mainstem bronchi), lymph nodes (mesenteric and cervical), ovaries with oviducts, parathyroid glands, pituitary gland, penis, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating glands, spinal column, spinal cord (cervical, thoracic, lumber), spleen, stomach, testes, thymus (where present), thyroid, trachea, urinary bladder, uterus-cervix and vagina.

HISTOPATHOLOGY: Yes, all organs that were included for fixation (5/sex of control and high dose group)

In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. A detailed qualitative evaluation of testes was performed on 5 randomly selected control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.

Other examinations:

Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test.
The criterion for statistical significance was p<0.05.

Clinical signs:

no effects observed

Description (incidence and severity):

No relevant clinical signs or mortality were observed in males and females throughout the study.

Mortality:

no mortality observed

Description (incidence):

No relevant clinical signs or mortality were observed in males and females throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences of toxicological significance were seen in terminal body weight or body weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No intergroup differences were seen in food consumption.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No changes of toxicological significance were observed. The statistically significant differences between control and treated animals (erythrocytes, mean corpuscular haemoglobin and leucocytes in males, haemoglobin, haematocrit and platelets in females) were of low magnitude and/or not dose-related, therefore considered incidental.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No changes of toxicological significance were observed. Statistically significant fluctuations of some biochemical parameters (mean group values) were recorded such as: increase of glucose in males dosed with 100 and 1000 mg/kg bw/day (48% for both dosages), increase in urea in those receiving 100 mg/kg bw/day (19%), increase of aspartate aminotransferases in females dosed with 300 mg/kg bw/day (35%), decrease of bilirubin (81%) and increase of potassium (10%) in those treated with 1000 mg/kg/day when compared to controls.
Due to the lack of dose- and sex-consistency and to the absence of other relevant findings, the above alterations were considered unrelated to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No significant differences were observed between groups, in motor activity, grip strength and sensory reactivity to stimuli, evaluated at the end of treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No relevant differences in organ weights were seen between the controls and treated animals of both sexes.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No remarkable changes were noted at post mortem examination in treated animals when compared with controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed. The lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the experimental conditions.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Spermatogenic cycle
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted. No relevant difference in estrous cycle was observed in treated females when compared to controls.

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:

other: no adverse effects observed

Key result

Critical effects observed:

no

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18 Dec 2012 - 29 Jan 2013

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

Limit test:

no

Species:

rat

Strain:

other: Wistar Hannover RccHan

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Models, S.L., Barcelona, Spain
- Age at study initiation: 11-12 weeks
- Mean weight at study initiation: males: 312 to 364 g; females: 198 to 219 g
- Fasting period before study: no
- Housing: cages with standard, granulated, softwood Lignocel S8/15 bedding
Pretreatment period: 5 per cage
Mating period: 1 male and 1 female per cage
Postmating: single housing
- Diet: pelleted standard Harlan Teklad 2014C rat/mouse maintenance diet; ad libitum
- Water: tap water; ad libitum
(Analyses of diet and water was performed.)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 30-60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 1% methylcellulose+1% Tween 80 in aqueous solution

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of dose formulation was determined in samples taken from the formulation to be administered to Groups 1 to 4 at the start and end of treatment by GC-FID analysis.

Duration of treatment / exposure:

Males: two weeks prior to mating and at least up to and including the day before sacrifice (day 49 of treatment).
Females: two weeks prior to mating and at least up to and including the day before sacrifice (day 4 postpartum).

Frequency of treatment:

Once daily; 7 days/week

Dose / conc.:

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

Dose / conc.:

300 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: The dose levels were selected based on a dose-range finding toxicity study in rats (Harlan Laboratories Study S40412 14-day Oral (gavage) DRF using dose levels of 100, 500 and 1000 mg/kg bw/day.

Observations and examinations performed and frequency:

MORTALITY AND CLINICAL SIGNS
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 2 hours after dosing
- Detailed clinical observations were performed on all test and control group animals before the first exposure to the test item and once weekly thereafter, at least two hours after dosing. Observations were also performed on females with litters on Day 4 postpartum. These observations were performed outside the home cage, in a standard arena, at least two hours after dosing (where applicable) to ensure that any transient effects of treatment are identified.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: twice weekly during the pre-pairing and pairing period and daily during postpairing period.
F0 females: twice weekly during the pre-pairing and pairing period and daily until day 4-5 postpartum.

FOOD CONSUMPTION:
F0 males: once weekly during the pre-pairing period and two weeks of postpairing period.
F0 females: once weekly during the pre-pairing period and days 0-7, 7-14, 14-21 postcoitum and days 1-4 postpartum.
No food consumption was recorded during the mating period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
F0 males: one week during the pre-pairing and postpairing period.
F0 females: one week during the pre-pairing, postcoitum period and days 1-4 postpartum.
No water consumption was recorded during the remaining periods.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Males: on day 50
Females: on day 5 postpartum
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters: haemoglobin, erythrocytes, leucocytes, differential blood count (absolute/relative), reticulocytes (count and maturity index), platelets, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Males: on day 50
Females: on day 5 postpartum
- Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin, cholesterol (total), creatinine, glucose, urea, total protein, calcium, chlorid, potassium, sodium, alanine aminotransferase (ALAT), alkaline phosphatase (AP), aspartate aminotransferase (ASAT), triglycerides, creatine kinase, gamma-glutamyl-transferase (gGT), inorganic phosphorus,

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Males: the day before sacrifice and at least two hours after dosing.
Females: on day 4 postpartum.
- Dose groups that were examined: all dose groups (5 animals/group)
- Battery of functions tested:
Functional performance test: Grip strength (fore- and hind limbs) and motor activity
Sensory reactivity assessment: Sensory reactivity to different stimuli (e.g. auditory, visual and proprioceptive)

-OTHER: spermatology stage evaluation

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (males on day 50, females on day 5 postpartum)
- Organ weights: epididymes and testicles, adrenal gland, brain, heart, kidney, liver, ovaries, pituitary, spleen, thymus, thyroid and parathyroids, uterus and oviducts
- Fixation: Adrenals, Aorta (thoracic), Bone with bone marrow- (Sternum), Peyer’s patches, Pharynx, Pituitary, Bone marrow smear (femur) (air-dried smear), Brain (cerebrum, cerebellum, medulla/pons), Epididymides (Bouin's solution), Esophagus, Eyes with optic nerve (Davidson’s fixative), Femur (with articular surface), Heart (with papillary muscle), Intestine, large (cecum, colon and rectum), Intestine, small (duodenum, jejunum and
ileum), Kidneys, Larynx, Liver, Lungs, with main bronchi and bronchioles, Lymph nodes (mandibular and mesenteric), Nose (the entire head will be collected), Mammary gland area, Ovaries, Pancreas, Prostate, coagulating gland and seminal vesicles, Salivary glands (mandibular, sublingual and parotid), Sciatic nerve, Skeletal muscle, Skin (abdominal), Spinal cord (cervical, thoracic and lumbar), Spleen, Stomach (glandular and nonglandular), Testes (Bouin's solution), Thymus, Thyroid (incl. parathyroid gland, if possible), Tongue, Trachea, Urinary bladder, Uterus (cervix, corpus and oviducts), Vagina, All gross lesions

HISTOPATHOLOGY: Yes, all organs that were included for fixation (5/sex of control and high dose group)

Statistics:

The following statistical methods were used to analyse food consumption during postpairing period, body weight, clinical laboratory data, organ weights, postnatal development and reproduction data, as well as macroscopic findings:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data cannot be assumed to follow a normal distribution.
- Fisher's exact-test was applied to the macroscopic findings.
- Armitage/Cochran Trend Test [5] for non-neoplastic lesions, if appropriate.
- Bonferroni-test was applied to some reproduction parameters.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant clinical signs were observed in males and females. One male and female from the control group and three males and one female at 100 mg/kg showed occasionally up of the product
(presumably immediate reaction to the administration of test substance). In addition, male No. 28 at 300 mg/kg bw/day had missing upper incisors in week 4 of treatment. No clinical signs were observed in females at 300 or 1000 mg/kg, while in males no clinical signs were oberved at 1000 mg/kg bw/day.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test-item-related differences from the control group were recorded. Some significant differences were observed at 1000 mg/kg bw/day but these were not considered of toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was similar in all groups.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Slight increase in water consumption was recorded in females at 300 and 1000 mg/kg bw/day during pregnancy period. These differences were not statistically significant. In males no differences from the control group were recorded.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No changes of toxicological relevance were recorded.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No changes of toxicological relevance were recorded in females. Bilirubin, cholesterol and triglyceride values tended to be higher at 300 and 1000 mg/kg bw/day in females. An increase in bile acid values was observed at 300 mg/kg bw/day in males (non adverse). The differences recorded were statistically significant with respect to the control group.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No differences in fore- or hind limb grip strength were recorded in males and females. Slightly lower locomotor activity was recorded in males at 100 mg/kg bw/day, being it statistically significant at 15 minutes. Slightly lower locomotor activity was recorded in females at 300 and 1000 mg/kg bw/day for 15-30 min, but the differences were not statistically significant. No differences were recorded in the remaining groups. All animals responded positively to the different reflexes such as blink, pinna, righting and iridic reflexes and pain, auditory startle and handling response (push-off).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, kidney weights tended to be slightly higher in males. These differences were not statistically significant. Moreover, lower pituitary and adrenal weights were recorded; the differences were statistically significant for pituitary in males. A trend to higher liver weight was recorded in females at 1000 mg/kg bw/day, but the differences were not statistically significant.
Concerning reproductive organs, lower left ovary weights were recorded at 1000 mg/kg bw/day (non adverse). This difference was statistically significant in relation to body weight ratio.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, large right kidney was observed in one male and reddish thymus in another one. A reddish area on cecum wall was observed in one male and reddish foci in lungs in another one. Likewise, thickened gastric mucosa was observed in one female and small spleen in another one. At 300 mg/kg bw/day, reddish foci were observed in the thymus of one male. No findings were observed in females. At 100 mg/kg bw/day, pale kidneys were observed in one male and reddish foci in lungs in another one. Likewise, left seminal vesicle reduced in size was observed in one male and reddish thymus in three males. Thickened gastric mucosa was observed in one female.
In the control group, one male had left seminal vesicle reduced in size. One male and one female had pale kidneys. In addition, one male had pancreas reduced in size and thymus with reddish foci. Yellowish-whitish gastric mucosa was recorded in few females from all groups (including control group). All macroscopic findings recorded were considered to be within the range of normal background lesions that may be seen in rats of this strain and age and under the experimental conditions used in this study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All microscopic findings recorded were considered to be within the range of normal background lesions that may be seen in rats of this strain and age and under the experimental conditions used in this study.

Histopathological findings: neoplastic:

no effects observed

Other effects:

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:

other: no adverse effects observed

Key result

Critical effects observed:

no

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 Mar - 14 Apr 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Wistar Crl:(WI) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 185-208 g (males); 153-173 g (females)
- Fasting period before study: no
- Housing: the animals were housed in groups of 5 per sex per cage in stainless steel suspended cages with wire mesh floors. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages with sterilised sawdust (B.M.I. Helmond, the Netherlands) provided as bedding.
- Diet: standard pelleted laboratory animal diet (Carfil Quality BVBA, Oud-Turnhout, Belgium), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 (optimal temperature)
- Humidity (%): 50 (optimal humidity level)
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 Mar 1998 To: 14 Apr 1998

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing and placed on a magnetic stirrer during dosing. Adjustment was made for the specific gravity of test substance and vehicle. The solution was shown to be stable for at least 4 hours.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle choice was based on trial formulations performed at the test laboratory
- Concentration in vehicle: 10, 40 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

GC-analyses were performed to determine the concentration of the test material of all dosing solutions. The actual concentrations were shown to be 95-107% of the nominal concentration, as analysed in duplicate samples. The homogeneity of the low- and high concentration samples was measured in duplicate samples. The concentration as sampled at 10, 50 and 90% height was shown to be 97-106% of nominal values. A relative difference of -2.8 - 2.1% in the stability from t=o to t=4 hours was measured in duplicate samples.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days/week

Dose / conc.:

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

Remarks:

administered by gavage, calculated weekly according to body weight

Dose / conc.:

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

Remarks:

administered by gavage, calculated weekly according to body weight

Dose / conc.:

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

Remarks:

administered by gavage, calculated weekly according to body weight

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: a range-finding study was performed with 3 rats/sex/dose, administered 0, 50 200 and 1000 mg/kg bw/day for 5 days (report No. 227306). There was no mortality. No treatment-related clinical signs were observed, no effect on body weight or food consumption was noted, there were no macroscopic findings, and the liver and kidney weights were comparable to those of the control group. Therefore, the same dose levels were selected for the main study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were observed for mortality twice daily and for clinical signs once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on Day 8, 15, 22 and 28, detailed clinical observations were made outside the cage in a standard arena. Symptomes were recorded and graded according to a fixed scale.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded on Day 1, 8, 15, 22 and 28 of the study

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes; ether
- Animals fasted: Yes, overnight for a maximum of 20 hours, with free access to water
- How many animals: all the animals in the control and treatment groups
- Parameters checked: erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, red cell distribution width, total leucocyte count, differential leucocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes), prothrombin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy
- Animals fasted: Yes, overnight for a maximum of 20 hours, with free access to water
- How many animals: all the animals in the control and treatment groups
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, bilirubin, cholesterol, creatinine, glucose, urea, total protein, albumin, alkaline phosphatase, sodium, potassium, chloride, calcium, inorganic phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of the treatment
- Dose groups that were examined: the control group and all treatment groups
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength, activity test (based on hourly data per animal, using a computerised motor activity monitoring system)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All the animals in the control and treatment groups were necropsied and a description of all macroscopic abnormalities was recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution:
adrenal glands, aorta, brain, caecum, cervix, clitoral gland, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, female mammary glandarea, femur including joint, heart, ileum, jejunum, kidneys, larynx, lacrimal gland (exorbital), liver, lung (infused with formalin), lymph nodes (mandibular, mesenteric), nasopharynx, oesophagus, ovaries, pancreas, Peyer's patches (jejunum, ileum, if detectable), pituitary gland, preputial gland, prostate, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, sceletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid, tongue, trachea, urinary bladder, uterus, vagina, all gross lesions.

The following organ weights were recorded from the surviving animals on the scheduled day of necropsy: adrenal glands, brain,
epididymides, heart, kidneys, liver, spleen, testes, thymus.

HISTOPATHOLOGY: Yes. The organ and tissue samples were processed, embedded and cut at a thickness of 2.4 micrometers and stained with haematoxylin and eosin. For the control and 1000 mg/kg bw/day groups, the following organs were histologically examined: adrenal glands, aorta, brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lung (infused with formalin), lymph nodes (mandibular, mesenteric), oesophagus, ovaries, pancreas, Peyer's patches (jejunum, ileum, if detectable), pituitary gland, preputial gland, prostate, rectum, sciatic nerve, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid, trachea, urinary bladder, uterus. The organs and tissues that were preserved but not examined during histopathology did not show signs of toxicity or other effects. The observed gross lesions were examined in animals from all the groups.

Statistics:

The following statistical methods were used to analyse the data: Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnett test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed in any group. Salivation was observed on one or several days in 1/5 males and 1/5 females in the low-dose group, and 2/5 males and 1/5 females in the high-dose group. As there is no dose-response effect and no other clinical or neurological signs that indicate that the effect is treatment-related, this is considered to be an incidental finding, which is probably caused by the administration procedure. Incidental observations of alopecia, red staining, piloerection and scabs were made, involving one or several animals. These findings are considered to be within the expected range for rats of this age and strain and regularly occur during a subacute study.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant differences in body weight or body weight gain were observed between the control and treatment groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant differences in food consumption were observed between the control and treatment groups.

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):

In females of the high-dose group, a statistically significant increase in relative leucocyte level (app. 8%) and decrease in relative neutrophil level (app. 8%) was noted, compared with the control group values. These values are related to each other as a percentage of the total lymphocyte concentration, and a variation in one will necessarily affect one or several other specific lymphocyte values. As the changes are minimal, no effects were seen in males and no other haematological effects were noted, this effect may be treatment-related, but is considered not to be of toxicological relevance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males of the high-dose group, the level of calcium and chloride was significantly increased. As these increases were around 5% compared with the control group levels and no similar effects were observed in the female groups they are not considered be of toxicological relevance. The statistically significant change in bilirubin level in mid-dose males is considered to be incidental, as no effect was seen in the highest dose level and the mean value was the same in all male groups.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

1/5 females in the control group showed a statistically significant increase in motor activity. This is considered to be an incidental occurrence as no other animals exhibited the same behaviour.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in absolute or relative organ weight in the treatment groups, compared to the control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

In 2/5 females in the mid-dose group watery fluid was observed in the uterus. This is a normal finding during a part of the estrus cycle in rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The incidence and severity of the observed lesions were similar in the control group and treatment groups and are considered to have occured spontaneously.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

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:

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

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Table 1: Clinical signs; salivation observed as number of rats per group and days per rat

Group (mg/kg bw/day)	Control	50	200	1000
Males (No. - days observed)	0	1 (Day 9)	0	2 (Day 9 and Day 9, 27-28)
Females	0	1 (Day 27-28)	0	2 (both Day 26-28)

Table 2: Haematology results

	Group (mg/kg bw/day)
	Males				Females
	Control	50	200	1000	Control	50	200	1000
Neutrophils (1)^1	0.093 ± 0.022	0.108 ± 0.049	0.116 ± 0.021	0.090 ± 0.047	0.138 ± 0.022	0.079 ± 0.031	0.115 ± 0.023	0.063 ± 0.018*
Lymphocytes (1)^1	0.883 ± 0.026	0.862 ± 0.053	0.851 ± 0.040	0.872 ± 0.056	0.830 ± 0.027	0.891 ± 0.027	0.851 ± 0.029	0.908 ± 0.027*
WBC (G/L)	13.5 ± 3.2	12.5 ± 0.5	12.1 ± 2.4	13.6 ± 2.7	7.7 ± 1.9	8.0 ± 1.8	8.2 ± 3.6	7.8 ± 1.9

*Statistically significant (p < 0.05)

¹relative part of total =1

 

Table 3: Clinical chemistry results

	Group (mg/kg bw/day)
	Males				Females
	Control	50	200	1000	Control	50	200	1000
Calcium (mmol/L)	2.53 ± 0.04	2.52 ± 0.04	2.52 ± 0.02	2.44 ± 0.02*	2.49 ± 0.07	2.47 ± 0.06	2.52 ± 0.05	2.47 ± 0.05
Chloride (mmol/L)	101 ± 2	100 ± 1	99 ± 2	97 ± 1**	98 ± 2	100 ± 2	100 ± 1`	98 ± 2

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

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:

The available information comprises adequate and reliable (Klimisch score 1) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to the endpoint discussion for further details). The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

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

Additional information

Read across justification

There are no data on the repeated dose toxicity of Hexadecyl palmitate (CAS 540-10-3). The assessment was therefore based on studies conducted with analogue substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Repeated dose toxicity, oral, subacute

CAS 22393-85-7

A combined repeated dose toxicity and reproduction/developmental toxicity screening study was performed according to OECD Guideline 422 and under GLP conditions (key study, 2014) with Tetradecyl oleate (CAS 22393-85-7). 10 rats/sex/dose were administered 0, 100, 300 and 1000 mg/kg bw/day once daily for 28-29 days (males) and up to 54 days (females) via oral gavage. The application started two weeks before mating on test day one and ended on the day of or one day before sacrifice. Day of sacrifice was on test day 29 or 30 for the male rats and on lactation day 3 or shortly thereafter for the female rats. There was no mortality during the study period. No toxicologically relevant clinical signs were observed. The body weight, body weight gain and food consumption was comparable between the control and treatment groups. There were no toxicologically relevant effects on organ weights. No changes of toxicological significance were observed for haematology and clinical chemistry parameters. There was no significant difference between control and treatment groups during the observational and neurological screenings. The macroscopic inspection at autopsy and subsequent histopathological examination did not reveal any treatment-related changes. The NOAEL for systemic toxicity was considered to be ≥ 1000 mg/kg bw/day for male and female rats.

 

CAS 93803-87-3

A 28-day oral repeated dose toxicity study was performed with octyldodecyl isooctadecanoate (CAS 93803-87-3) according to OECD 407 and under GLP conditions (supporting study, 1998). Five Wistar rats/sex/dose were administered 0, 50, 200 and 1000 mg/kg bw/day by gavage, for 28 consecutive days. There was no mortality and no toxicologically relevant clinical signs were observed during the study period. No significant differences in body weight, body weight gain and food consumption between the control group and treatment groups were noted. In females of the high-dose group, a statistically significant increase in relative leucocyte level (approx. 8%) and decrease in relative neutrophil level (approx. 8%) was noted, compared with the control group values. These values are related to each other as a percentage of the total lymphocyte concentration, and a variation in one will necessarily affect one or several other specific lymphocyte values. As the changes are minimal, no effects were seen in males and no other haematological effects were noted, this result is considered not to have toxicological relevance. In males of the high-dose group, the level of calcium and chloride was significantly increased, compared with the control group. These increases were around 5% compared with the control group levels and no similar effects were observed in the female groups. Therefore, they are not considered to have toxicological relevance. No treatment-related effects on neurobehavioral parameters were observed. There were no significant differences in absolute or relative organ weight in the treatment groups, compared with the control group. The macroscopic inspection at autopsy and subsequent histopathological examination did not reveal any treatment-related changes. The NOAEL for systemic toxicity was considered to be ≥ 1000 mg/kg bw/day for male and female rats.

CAS 17671-27-1

A combined repeated dose toxicity and reproduction/developmental toxicity screening study was performed with Docosyl docosanoate (CAS 17671-27-1) according to OECD guideline 422 and under GLP conditions (supporting study, 2014). Ten rats/sex/dose were administered 0, 100, 300 and 1000 mg/kg bw/day Docosyl docosanoate once daily via gavage up to day 49 of treatment (males) or day 4 postpartum (females). The application started two weeks before mating on test day one and ended one day before sacrifice. No test item-related premature death was noted. No test item-related signs of toxicity were noted during the observational and neurological screenings. A treatment-related decrease in body weight was noted for the female animals of the high dose group (1000 mg /kg bw/day) on lactation day 4. No toxicologically relevant effects were observed on the haematological and clinical chemistry parameters. The macroscopic inspection at autopsy and subsequent histopathological examination did not show any treatment-related changes. Based on the absence of adverse effects the NOAEL for systemic toxicity was considered to be ≥ 1000 mg/kg bw/day for male and female rats.

Overall conclusion for repeated dose toxicity

The data for the read-across analogue substance showed that no effects were observed up to and including the recommended limit values. Therefore, as the available data did not identify any hazard for repeated dose toxicity, the target substance Hexadecyl palmitate (CAS 540-10-3) is not expected to be hazardous following repeated exposure.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Hexadecyl palmitate (CAS 540-10-3), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on repeated dose toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.