Sorbitan, tridocosanoate

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:

05 Jan - 18 Aug 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Jan - 18 Aug 2021

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:

adopted in 2016

Deviations:

yes

Remarks:

5/10 animals/sex of the control and high dose group were selected for recovery whereas the guideline recommends to increase the number of animals to 5 additional rats/sex for each recovery group; no details on terminal procedures with pups provided

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Hino Breeding Center, Japan
- Age at study initiation: 9 weeks
- Weight at study initiation: 190 - 270 g (males) and 140 - 210 g (females)
- Housing: In groups of 2-3/sex/cage in hanging stainless steel cages with wire-mesh floor cages measuring 260 x 380 x 180 mm for quarantine and acclimatisation. After group allocation the animals were housed individually in hanging stainless steel cages with wire-mesh floor cages measuring 260 x 380 x 180 mm and 165 x 300 x 150 mm. Irradiated hemp mats with gamma ray were used for enrichment. During mating the females were housed in the males cages (1:1). After mating, females were housed in polycarbonat cages measuring 265 x 426 x 150 mm with wooden bedding and autoclaved gnawing wood enrichment.
- Diet: Pelleted diet, MF (Oriental Yeast), ad libitum
- Water: Chlorinated tap water from the municipal water supply (3-5 ppm level), administered via an automatic watering system for hanging cages and in polycarbonate bottles for polycarbonate cages, ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:
Food and water quality were confirmed by analytical methods at regular intervals in the testing facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 Jan 2021 To: 05 May 2021

Route of administration:

oral: gavage

Vehicle:

other: 5% w/v gum arabic solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed and gum arabic was added at final concentration of 5% w/v. Purified water was added to this mixture and kneaded to prepare 10.0% w/v formulation. A part of the 10.0% w/v formulation was taken with stirring by a magnetic stirrer and diluted with 5% w/v gum arabic solution to prepare the 1.00 and 3.00% w/v formulations.
The formulations and vehicle were subdivided into plastic containers and stored at the cold place (target range: 1 to 10 °C). On each dosing day formulations and vehicle were taken out from the storage place and dosed to the animals. The formulations were used within a stable period of 16 days after preparation


VEHICLE
- Justification for use and choice of vehicle: The test material did not dissolve in purified water or olive oil, but was found to be homogenate with 5% w/v gum arabic solution at a concentration of 10% w/v (non-GLP). The vehicle was used for a general toxicity and historical control data are available.
- Concentration in vehicle: 1, 3 and 10 % w/v
- Amount of vehicle: 20 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the test item formulations at 10.0 and 1.00 w/v% were confirmed with high performance liquid chromatography (HPLC) as part of a different study (CERI study no. X02-0328) from the bottom, middle and upper part of the formulations. The coefficient of variations of the concentrations of each layer were found to be in an acceptable range and all formulations were confirmed to be homogenious. Dose formulations were confirmed to be stable after 16 days of storage in a cold place.

Concentrations of the test item formulations were confirmed with HPLC in the first preparation (CERI study no. X02-0328). Actual concentrations of 10.0, 3.00 and 1.00% w/v formulations were 10.2, 3.06 and 1.03% w/v, respectively and considered to be acceptable.

Duration of treatment / exposure:

Males were treated daily for 29 days, starting from 14 days prior to mating and including the mating period.
Females that delivered were treated daily for 14 days prior to mating, through mating and gestation until lactation Day 13.
Non-mating females of the satellite group were treated for 29 days.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

Not applicable for an OECD 422 study

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:

Main group: 5 (control and high-dose group) and 10 (low- and mid-dose group)
Non-mating control: 10 (control and high-dose group only)
Recovery group: 10 (each 5 from the main group and 5 from the non-mating control; control and high-dose group only)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on the results of a 14-day repeated dose oral toxicity study (CERI study no. C21-0051). Groups of 3 rats/sex were administered dose levels of 50, 200, 500 and 1000 mg/kg bw/day. No clinical signs of toxicity were noted and all animals gained weight as expected. Necropsy and organ weights of the liver, kidneys and spleen revealed no abnormal findings. The top dose for the main study was therefore set to 1000 mg/kg bw/day.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for clinical signs and mortality during the treatment period and once daily during the recovery period. Females were observed for delivery and nursing conditions.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made prior to dosing and once weekly thereafter.
- Examinations included: Animal reactions for external stimuli, handling observations and arena observations.
Animal reactions for external stimuli: Holding animals or bringing hand close to hold, easiness of removal and vocalisation
Handling observations: Muscle tone, subnormal temperature, hair appearance including piloerection, stained hair and unkempt hair, skin and mucous colour such as paleness, reddening and cyanosis, eyes including lacrimation, exophthalmos and pupillary size, salivation and secretion
Arena observations: Posture, motor activity level, respiration, gait characteristics, lid closure, tremor, twitch, convulsion, stereotypical behaviour and abnormal behaviour for one min or more (within five min), frequencies of defecation (number of faeces) and urination (number of pools)

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded for males and non-mating females on Days 1, 3, 7, 14, 21 and 28 and on Days 1, 7 and 14 of recovery and at necropsy. For females, individual body weights were recorded on Days 1, 3, 7 and 14, on gestation Days 0, 7, 14 and 20 and on postnatal days 0, 4, 8 and 13. For non-copulated females, body weight was recorded on Days 21, 28, 35, 42 and 49.

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

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: Blood was collected at terminal sacrifice from the abdominal aorta.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/sex from the main group, 5 females from the non-mating group and males and females of the recovery group.
- Parameters examined: Red blood cell count (RBC), haemoglobin (Hb), haematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (Platelet), reticulocyte count ratio (Reticulo), white blood cell count (WBC), neutrophils (Neutro), lymphocytes (Lymph), eosinophils (Eosino), basophils (Baso) and monocytes (Mono), prothrombin time (PT) and activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected at terminal sacrifice from the abdominal aorta.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/sex from the main group, 5 females from the non-mating group and males and females of the recovery group.
- Parameters examined: Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (g-GTP), total cholesterol (T-Cho), triglycerides (TG), blood urea nitrogen (BUN), creatinine, total protein (T-Protein), albumin, A/G ratio, glucose, total bilirubin (T-Bil), total bile acids (TBA), inorganic phosphorus (IP), calcium (Ca), sodium (Na), potassium (K) and chloride (Cl)

PLASMA/SERUM HORMONES/LIPIDS: Yes
Blood was collected at terminal sacrifice from the abdominal aorta.
- Animals fasted: Yes
- How many animals: 5 males from the main group and 5 males from the recovery group.
- Parameters examined: Thyroid hormone (T4)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from each 5 animals/sex/group from the afternoon of the last observation day until the next morning, covering a period of 15-17 h.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Urine volume, colour, turbidity, specific gravity, pH, protein, glucose, occult blood and urinary sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In each last dosing week.
- Dose groups that were examined: 5 animals/sex from each group (main group, non-mating group and recovery group).
- Battery of functions tested: Sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

Vaginal smears of all females in the mating group were collected from Day 1 to 14 of dosing. The stages of oestrous cycle were determined with a light microscope after giemsa staining. The days from oestrus to the next oestrus were regarded as an oestrous cycle length and the mean oestrous cycle length was calculated. When the oestrus was successive the first day was regarded as an oestrus.

Sperm parameters (parental animals):

The male gonads were histopathologically examined.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
One or two pups per litter were sacrificed on Day 4 after birth.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
Not specified

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: On PND 4 and 13
- Animals fasted: No
- How many animals:
On PND 4: 1-2 pups/litter
On PND 13: 2 pups/litter
- Parameters examined: Thyroid hormone levels (T4)


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes

All parental animals were subjected to a detailed gross necropsy including external surface of the body, all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities, and their contents after bleeding from the ventral aorta under isoflurane anesthesia on the next day of last dosing and last recovery days. Non-copulated female, non-delivered females and dead female were subjected as with a survived animal on each found day. Vaginal smears were collected before gross necropsy in survived females, and stages of the oestrous cycle were determined with a light microscope after Giemsa staining. The number of implantation sites of the uterus after incision were counted for the mated females. Organs and tissues were collected from all animals of all groups. Trachea, lungs and urinary bladder were inflated with 10% neutralized buffered formalin before removal. Stomach and intestines were filled and fixed with 10% neutralized buffered formalin and the contents were washed away with water.
The following organ weights were recorded: Liver, heart, kidneys, testes, epididymides, prostate (with a part of the urethra), seminal vesicles (with coagulating gland), ovaries, uterus, brain, spleen, thymus, thyroid (with parathyroid) and adrenals.


HISTOPATHOLOGY: Yes
All organs/tissues were preserved in 10% neutralized buffered formalin. Testes and epididymides were fixed in modified Davidson’s fixative. Decalcification was done for the bone and bone marrow (femur) with 10% formic acid formalin before trimming.
The following organs and tissues were examined:
Respiratory system: Trachea, lungs
Digestive system: Submandibular glands, stomach, intestines (duodenum to rectum, including Peyer's patches), pancreas, liver
Cardiovascular system: Heart
Urinary system: Kidneys, urinary bladder
Reproductive system: Testes, epididymides, prostate (ventral and dorsolateral lobes), seminal vesicles (including coagulating gland), ovaries, uterus (horn and cervix), vagina
Nervous system: Brain (including cerebrum, cerebellum and pons), spinal cord (thoracic) and sciatic nerve
Hematopoetic system: Bone marrow (femur), axillar lymph nodes, mesenteric lymph nodes, spleen and thymus
Endocrine system: Pituitary gland, thyroid (including parathyroid) and adrenals
Sense organ: Eyeballs
Musculoskeletal system: Skeletal muscle (femoral region), bone (femur)
Skin and acessory organ: Mammary gland

Light microscopic examinations were performed after embedding in paraffin, sectioning and hematoxylin and eosin (HE) staining in each 5 animals of the control and high dose groups. Spleen in females of the recovery group was examined since treatment-related changes were suspected in organ weight. All macroscopic lesions were examined.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 13 days of age.

Statistics:

Data on body weights of parental animals, food consumption, grip strength, locomotor activity count, haematology and blood chemistry, urine volume, urine specific gravity, organ weights, body weights on necropsy, mean oestrous cycle length, pairing days until copulation, gestation length, number of implantation sites, number of pups born, number of live pups, body weights of pups, AGD, number of nipples/areolae and T4 level of pups were analysed by Bartlett’s test for homogeneity of variance. If significant difference was not noted, the values of the control group and each test item group were analysed by Dunnet’s test. If significant difference was noted in the Bartlett test, the nonparametric Dunnet’s test was performed. The frequencies of defecation and urination were analysed by nonparametric Dunnett’s test. Body weights of pups were calculated on each sex as sample unit for each litter. Abnormal oestrous cyclicity, copulation index, conception index and delivery dam index were analysed by Fisher’s exact test between the control group and each test item group.
Indexes of delivery, birth, viability and sex ratio were examined by Bartlett’s test. If significant difference was not noted, the values of the control group and each test item group were analysed by Dunnet’s test. If significant difference was noted, the nonparametric Dunnet’s test was performed.
Data regarding body weights and food consumption during the recovery period, and parameters of haematology and blood chemistry, urine volume, urine specific gravity, organ weights and body weights at necropsy day for the recovery group were analysed by F-test for variance ratio. If there were no significant differences at a significance level of 5%, the Student t-test was performed. If there were significant differences at a significance level of 5% in the F-test, the Aspin-Welch test was performed. The frequencies of defecation and urination during the recovery period were analysed by Mann-Whitney U-test.

Reproductive indices:

Copulation index (%) = (Number of copulated pairs / Number if mated pairs) x 100
Conception index (%) = (Number of pregnant females / Number of copulated pairs) x 100
Delivery dam index (%) = (Number of pregnant females with live pups / Number of pregnant females) x 100
Delivery index (%) = (Number of pups born / Number of implantation sites) x 100

Offspring viability indices:

Birth index (%) = (Number of live pups at birth / Number of implantation sites) x 100
Viability index at birth (%) = (Number if live pups at birth / Number of pups at birth) x 100
Sex ratio of pups at birth = Number of live males at birth / Number of live pups at birth
Viability index on Day 4 after birth (%) = (Number of live pups on Day 4 / Number if live pups at birth) x 100
Sex ratio of pups on Day 4 after birth = Number of live males on Day 4 / Number of live pups on Day 4

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, 1/10 females showed staining around the nose and mouth on the day of delivery. At 300 mg/kg bw/day, 1/10 animals showed incomplete treatment of placenta and died during delivery. The effects were not related to treatment. There were no findings in males.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no mortality at 1000 mg/kg bw/day. At 100 and 300 mg/kg bw/day, each 1/10 females died during delivery. The deaths were not related to treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant decrease in food consumption noted in males at 1000 mg/kg bw/day on Day 7 of the recovery period. As no findings were observed during the treatment-period and there were no related changes in body weight, the decrease was considered toxicologically not significant. For details, please refer to Table 1 under "Any other information on results incl. tables".

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):

In males at 100 mg/kg bw/day, MCHC was decreased (-2%). The finding was without any dose-response relationship and therefore not attributed to treatment.
There were no findings in mating females. In non-mating females, there was an increase in neutrophils (+60%) and a decrease in lymphocytes (-8%) at 1000 mg/kg bw/day. The findings were within the laboratory’s historical control range and, in the absence of any related changes, considered to be incidental.
After the recovery period, males of the 1000 mg/kg bw/day group showed an increase in reticulocytes (+20%), and females of the 1000 mg/kg bw/day group showed an elongated APTT (+1.5 s). The findings were not observed during the treatment period and/or within the laboratory’s historical control data and therefore not related to treatment.
For details, please refer to Table 2 under “Any other information on results incl. tables”.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant increase in chloride concentration in non-mating females at 1000 mg/kg bw/day (+2%). The finding fell within the laboratory’s historical control range and there were no related findings, therefore the change was considered to be incidental. For details, please refer to Table 2 under “Any other information on results incl. tables”.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: T4 hormone levels, epididymides weight and histopathology, oestrus cyclicity, liver weight, mammary gland histopathology, ovary weight and histopathology, prostate weight and histopathology, seminal vesicles (with coagulating glands) weight and histopathology, testes weight and histopathology, thyroid weight and histopathology, uterus (with cervix) weight and histopathology, vagina histopathology, vaginal smears, adrenals weight and histopathology, brain weight and pituitary histopathology, fertility, foetal development, gestation length, litter size, litter viability, litter/pup weight, number of implantations and corpora lutea, number of live births, pre- and post-implantation losses, presence of anomalies, reproduction and sex ratio.
For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, 1/10 males showed epithelial cells in the urine. In non-mating females, cloudy urine and white blood cells were observed at 0 and 1000 mg/kg bw/day without dose-relationship after treatment and after recovery. Epithelial cells were observed in the control group only. The findings were not related to treatment.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, females showed decreased motor activity in the 50-60 min interval. The change was considered as single occurrence since no abnormal changes were observed in total (0-60 min interval) and there were no related changes in other parameters. There were no findings at 100 or 300 mg/kg bw/day. For details, please refer to Table 3 under "Any other information on results incl. tables".

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related findings. Any observations occurred sporadically in control and test item-treated animals. The dead female at 100 mg/kg bw/day had staining of the lower abdomen, dark reddish change and oedematous change of the lungs, perforation in the forestomach and blackish region of the mucosa of the glandular stomach. The dead female at 300 mg/kg bw/day showed staining in the lower abdomen, dark reddish change and oedematous change of the lungs, bilateral discoloration if the kidneys, small thymus, bilateral enlargement of the adrenals and hydrothorax.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone levels (T4) in males:
T4 levels were not affected by treatment at any dose level.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

1/10 females of the control group showed abnormal oestrus cycle. The finding was considered to be incidental.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Copulation was not confirmed in 1/10 control animals, and this female delivered her pups and pregnancy was confirmed. Copulation and pregnancy were not confirmed in 1/10 animals at 100 mg/kg bw/day and pregnancy was not confirmed in 1/10 animals at 1000 mg/kg bw/day. These changes were considered to be single occurrences since a dose-relationship was not noted.
Copulation index, conception index, number of implantations, gestation length, delivery dam index and delivery index were not affected in any test item group. At 100 mg/kg bw/day, the delivery index was increased when compared to control animals. Without a dose-response relationship, the finding was considered to be incidental.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

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

Dose descriptor:

NOAEL

Remarks:

fertility

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

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related deaths. The number of dead pups in the respective dose groups was 1/128, 6/122, 3/125 an 4/119 at 0, 100, 300 and 1000 mg/kg bw/day.
The number of pups (+21%) and the number of live pups (+20%) on Post-Natal Day (PND) 0 was slightly increased at 100 mg/kg bw/day. In the absence of a dose-response relationship, the finding was considered to be incidental. Birth index, viability index at birth and on PND 4 were not affected by treatment.
For details on reproductive and litter data, please refer to Table 6 under "Any other information on results incl. tables".

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blackish change of the tail tip was observed in one pup of the control group. Subnormal temperature and no milk band were observed in 16 pups at 300 mg/kg bw/day. The findings were not attributed to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis (T4 levels): There were no treatment-related findings.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

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

Key result

Reproductive effects observed:

no

Table 1: Summary of food consumption data in males (g/rat/day)

	Males
Dose (mg/kg bw/day)	0	100	300	1000
Group	Main + Recovery	Main	Main	Main + Recovery
Day 3	26.23 ± 1.68	26.80 ± 1.59	27.19 ± 1.89	27.18 ± 0.68
Day 7	26.31 ± 1.58	27.40 ± 2.27	27.39 ± 1.65	26.96 ± 1.46
Day 14	25.95 ± 1.28	26.65 ± 1.88	27.03 ± 1.82	26.23 ± 1.06
Day 28	26.16 ± 1.22	27.51 ± 1.50	27.08 ± 1.87	26.21 ± 1.77
Recovery period Day 7	26.62 ± 1.29	-	-	24.94 ± 0.90*
Recovery period Day 14	26.34 ± 2.16	-	-	26.20 ± 1.87
* significantly different from control at p < 0.05

Table 2: Summary of findings in haematology and clinical chemistry findings

Males

Females

Main group

Recovery group

Main group (Mating)

Non-mating

Recovery group (Mating)

Dose (mg/kg bw/day)

0

100

300

1000

0

1000

0

100

300

1000

0

1000

0

1000

Number of animals

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Haematology parameters

MCHC (g/dL)

36.44 ± 0.13

35.80 ± 0.51*

35.90 ± 0.38

36.32 ± 0.41

36.86 ± 0.51

36.52 ± 0.81

36.58 ± 0.54

36.60 ± 0.43

35.68 ± 0.78

36.18 ± 0.53

36.30 ± 0.46

36.34 ± 0.68

36.16 ± 0.49

36.18 ± 0.73

Reticulo (%)

4.034 ± 0.321

4.038 ± 0.533

3.888 ± 0.999

3.686 ± 0.375

3.302 ± 0.260

3.954 ± 0.354*

4.056 ± 1.321

3.286 ± 0.741

4.750 ± 2.303

4.258 ± 0.707

3.696 ± 0.550

3.756 ± 0.741

3.572 ± 0.371

3.980 ± 0.375

HCD mean Range: mean ± 2 SD

3.079 2.26 - 3.72 2.267 - 3.891

not reported

Neutro (%)

19.34 ± 7.26

17.10 ± 4.87

22.32 ± 5.41

18.52 ± 1.35

16.98 ± 1.19

21.18 ± 5.56

44.70 ± 6.44

45.32 ± 4.56

39.42 ± 7.82

37.48 ± 8.71

11.78 ± 4.45

18.84 ± 2.49*

18.48 ± 3.91

16.66 ± 6.48

HCD mean Range: mean ± 2 SD

not reported

20.49 11.4 - 36.2 3.95 - 37.03

Lymph (%)

74.72 ± 7.83

76.48 ± 5.26

71.70 ± 5.78

75.24 ± 1.20

75.60 ± 2.63

71.58 ± 6.79

48.84 ± 6.64

47.18 ± 4.83

53.92 ± 6.77

55.30 ± 7.25

83.22 ± 4.68

76.44 ± 2.77*

75.66 ± 4.90

78.00 ± 6.60

HCD mean Range: mean ± 2 SD

not reported

74.31 58.2 - 84.2 57.25 - 91.37

APTT (sec)

24.80 ± 2.63

25.10 ± 1.59

25.20 ± 1.62

24.48 ± 2.22

25.02 ± 1.19

24.86 ± 3.82

17.90 ± 0.57

18.44 ± 2.32

17.66 ± 1.61

17.24 ± 1.66

19.68 ± 1.29

18.14 ± 2.11

15.38 ± 2.09

19.40 ± 1.08**

HCD mean Range: mean ± 2 SD

not reported

16.63 9.4 - 18.9 12.37 - 20.89

Clinical chemistry parameters

Chloride (mEq/L)

106.22 ± 1.08

105.56 ± 0.83

106.10 ± 0.88

105.42 ± 0.46

105.18 ± 1.18

104.74 ± 1.13

100.60 ± 3.12

101.18 ± 2.14

102.28 ± 2.49

101.00 ± 2.37

106.42 ± 1.36

108.96 ± 1.00**

106.72 ± 1.25

105.84 ± 0.89

HCD mean Range: mean ± 2 SD

not reported

107.06 103.7 - 109.6 103.80 - 110.32

significantly different from control at * p < 0.05 and ** p < 0.01; MCHC: mean corpuscular haemoglobin concentration; Reticulo: reticulocytes; Neutro: neutrophils; Lymph: lymphocytes; APTT: activated partial thromboplastine time; HCD: historical control data

Table 3: Summary of findings on motor activity in males and non-mating females during Week 4 and in mating females during Week 7

	Males				Females
					Non-mating		Mating
Dose (mg/kg bw/day)	0	100	300	1000	0	1000	0	100	300	1000
Number of animals	5	5	5	5	5	5	5	5	5	5
0-10	45 ± 28	44 ± 36	30 ± 16	36 ± 6	74 ± 60	93 ± 21	48 ± 17	30 ± 21	30 ± 25	25 ± 31
10-20	19 ± 12	18 ± 11	16 ± 2	20 ± 10	29 ± 30	54 ± 30	36 ± 26	15 ± 13	9 ± 8	13 ± 15
20-30	8 ± 4	12 ± 14	18 ± 7	19 ± 10	25 ± 27	34 ± 24	13 ± 11	7 ± 11	20 ± 21	3 ± 4
30-40	7 ± 8	7 ± 6	5 ± 4	7 ± 10	8 ± 13	26 ± 21	22 ± 22	4 ± 5	7 ± 10	7 ± 11
40-50	9 ± 6	13 ± 13	6 ± 5	13 ± 14	3 ± 3	12 ± 18	3 ± 4	3 ± 5	5 ± 8	9 ± 18
50-60	2 ± 2	4 ± 6	7 ± 10	4 ± 3	7 ± 8	19 ± 15	32 ± 17	8 ± 11	13 ± 14	1 ± 1**
Total	91 ± 46	99 ± 63	83 ± 34	99 ± 36	145 ± 120	236 ± 67	153 ± 84	66 ± 23	84 ± 63	57 ± 53
**: significantly different from control at p < 0.01

Table 4: Organ weight findings

Males

Females

Main group

Recovery group

Main group (Mating)

Non-mating

Recovery group (Mating)

Dose (mg/kg bw/day)

0

100

300

1000

0

1000

0

100

300

1000

0

1000

0

1000

Number of animals

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Adrenals

Absolute weight (mg)

55.22 ± 3.53

69.81 ± 12.28*

64.75 ± 6.24

67.50 ± 12.82

62.02 ± 8.26

64.62 ± 7.09

73.21 ± 12.29

79.71 ± 10.86

84.44 ± 8.45*

74.57 ± 6.48

63.98 ± 13.00

70.92 ± 9.91

68.00 ± 7.07

68.68 ± 4.09

Relative weight (mg/100 g)

12.14 ± 1.15

14.97 ± 2.54*

13.87 ± 1.57

14.84 ± 2.36

12.58 ± 1.24

13.14 ± 1.75

24.59 ± 4.17

27.43 ± 3.17

28.27 ± 3.25

24.56 ± 2.07

24.78 ± 4.42

27.56 ± 3.33

24.32 ± 2.96

24.52 ± 2.84

Thymus

Absolute weight (mg)

504.76 ± 70.34

414.48 ± 75.77

450.27 ± 78.89

473.36 ± 41.05

387.86 ± 115.89

319.94 ± 82.94

198.35 ± 70.76

160.28 ± 36.57

182.62 ± 70.39

203.13 ± 39.22

411.42 ± 79.20

391.66 ± 98.01

348.94 ± 100.21

302.60 ± 78.16

Relative weight (mg/100 g)

110.26 ± 13.35

89.19 ± 17.03*

95.77 ± 12.61

104.34 ± 7.44

78.00 ± 18.93

65.00 ± 17.36

66.28 ± 21.98

55.40 ± 13.50

61.09 ± 23.42

67.06 ± 13.88

159.78 ± 28.74

151.66 ± 33.75

122.54 ± 24.70

106.24 ± 20.32

Spleen

Absolute weight (g)

0.690 ± 0.074

0.797 ± 0.106

0.812 ± 0.115

0.766 ± 0.186

0.762 ± 0.090

0.838 ± 0.124

0.498 ± 0.076

0.501 ± 0.049

0.531 ± 0.064

0.561 ± 0.083

0.516 ± 0.092

0.532 ± 0.071

0.612 ± 0.044

0.512 ± 0.081*

Relative weight (g/100 g)

0.152 ± 0.013

0.172 ± 0.022

0.172 ± 0.017

0.170 ± 0.042

0.156 ± 0.013

0.170 ± 0.030

0.166 ± 0.021

0.174 ± 0.018

0.179 ± 0.023

0.184 ± 0.026

0.200 ± 0.035

0.208 ± 0.033

0.220 ± 0.010

0.180 ± 0.025*

* significantly different from control at p < 0.05

Table 5: Gross necropsy findings in the glandular stomach

	Males						Females
	Main group				Recovery group		Main group (Mating)				Non-mating		Recovery group (Mating)
Dose (mg/kg bw/day)	0	100	300	1000	0	1000	0	100	300	1000	0	1000	0	1000
Number of animals examined	5	5	5	5	5	5	10	10	10	10	10	10	10	10
Glandular stomach
Blackish region of mucosa	0/5	0	0	0	0	0	5	4/0/1^#	4/0^$	3/0^§	1	0	0	0
Recessed region of mucosa	0	0	0	0	0	0	1	0/0/0^#	0/0^$	0/0^§	0	0	0	0
^#: scheduled sacrifice animal/ not copulated animal/ dead animal;^$: scheduled sacrifice animal/ dead animal;^§: scheduled sacrifice animal/ female animal did not deliver her pups

Table 6: Reproductive and litter data

Dose level (mg/kg bw/day)	0	100	300	1000
Number of pregnants	10	9	10	9
Gestation length (Mean ± S.D., day)	22.2 ± 0.4	22.3 ± 0.5	22.4 ± 0.5	22.2 ± 0.4
Number of implantation sites (Mean ± S.D.)	14.4 ± 1.6	14.6 ± 5.3	15.6 ± 1.8	13 ± 4.8
Delivery dam index (%)	100.0 (10/10)	88.9 (8/9)	90.0 (9/10)	100.0 (9/9)
Delivery index (Mean ± S.D., %)	89.3 ± 9.7	97.9 ± 4.2 *	91.9 ± 6.1	91.8 ± 6.4
Number of dams with live pups
on postpartum day 0	10	8	9	9
on postpartum day 4	10	8	9	9
PND 0
Number of pups (Mean ± S.D.)	12.9 ± 2.3	15.6 ± 0.7 *	14.3 ± 2.3	13.2 ± 1.2
Number of live pups (Mean ± S.D.)	12.8 ± 2.4	15.3 ± 0.9 *	13.9 ± 1.8	13.2 ± 1.2
Birth index (Mean ± S.D., %)	88.6 ± 11.1	95.6 ± 6.6	89.5 ± 8.4	91.8 ± 6.4
Viability index (Mean ± S.D., %)	99.0 ± 3.2	97.6 ± 3.3	97.5 ± 7.4	100.0 ± 0.0
Sex ratio of live pups (Mean ± S.D.)	0.39 ± 0.11	0.48 ± 0.08	0.54 ± 0.12 *	0.50 ± 0.11
Number of live pups with external anomalies	1/128	0/122	0/125	0/119
Pups weight (Mean ± S.D., g)
Male	7.09 ± 0.95	6.35 ± 0.33	6.75 ± 0.69	6.72 ± 0.61
Female	6.73 ± 0.85	6.13 ± 0.29	6.37 ± 0.79	6.37 ± 0.52
PND 4
Number of live pups (Mean ± S.D.)	12.7 ± 2.6	14.5 ± 1.3	13.8 ± 1.9	12.8 ± 1.9
Viability index (Mean ± S.D., %)	98.9 ± 3.5	95.1 ± 6.9	99.2 ± 2.4	96.6 ± 10.3
Sex ratio of live pups (Mean ± S.D.)	0.40 ±0.10	0.46 ± 0.09	0.54 ± 0.13 *	0.49 ± 0.10
Pups weight (Mean ± S.D., g)
Male	11.80 ± 1.89	10.51 ± 0.58	10.74 ± 2.08	10.78 ± 1.04
Female	11.25 ± 1.66	10.09 ± 0.54	10.27 ± 1.99	10.32 ± 1.00
AGD (AGD/3^ body weight)
Male	2.19 ± 0.14	2.17 ± 0.08	2.23 ± 0.16	2.2 ± 0.11
Female	1.08 ± 0.05	1.04 ± 0.03	1.14 ± 0.09	1.02 ± 0.05
PND 13
Number of live pups (Mean ± S.D.)	10.8 ± 2.4	12.5 ± 1.3	11.6 ±1.7	10.9 ± 1.6
Number of nipples/areolae in males (Mean ± S.D.)	0.0 ± 0.0	0.0 ± 0.1	0.0 ± 0.0	0.0 ± 0.1
Pups weight (Mean ± S.D., g)
Male	31.35 ± 4.49	28.70 ± 1.32	29.20 ± 4.44	30.14 ± 2.66
Female	30.31 ± 4.09	27.86 ± 1.22	27.99 ± 4.15	29.05 ± 2.58
* significantly different from control at p < 0.05

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity, fertility and developmental toxicity was established at 1000 mg/kg bw/day, the highest dose level tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

5/10 animals/sex of the control and high dose group were selected for recovery whereas the guideline recommends to increase the number of animals to 5 additional rats/sex for each recovery group; no details on terminal procedures with pups provided

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sorbitan, tridocosanoate

EC Number:

301-037-8

EC Name:

Sorbitan, tridocosanoate

Cas Number:

93980-59-7

Molecular formula:

C72H138O8

IUPAC Name:

Sorbitan tridocosanoate

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Hino Breeding Center, Japan
- Age at study initiation: 9 weeks
- Weight at study initiation: 190 - 270 g (males) and 140 - 210 g (females)
- Housing: In groups of 2-3/sex/cage in hanging stainless steel cages with wire-mesh floor cages measuring 260 x 380 x 180 mm for quarantine and acclimatisation. After group allocation the animals were housed individually in hanging stainless steel cages with wire-mesh floor cages measuring 260 x 380 x 180 mm and 165 x 300 x 150 mm. Irradiated hemp mats with gamma ray were used for enrichment. During mating the females were housed in the males cages (1:1). After mating, females were housed in polycarbonat cages measuring 265 x 426 x 150 mm with wooden bedding and autoclaved gnawing wood enrichment.
- Diet: Pelleted diet, MF (Oriental Yeast), ad libitum
- Water: Chlorinated tap water from the municipal water supply (3-5 ppm level), administered via an automatic watering system for hanging cages and in polycarbonate bottles for polycarbonate cages, ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: Food and water quality were confirmed by analytical methods at regular intervals in the testing facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 Jan 2021 To: 05 May 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 5% w/v gum arabic solution

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed and gum arabic was added at final concentration of 5% w/v. Purified water was added to this mixture and kneaded to prepare 10.0% w/v formulation. A part of the 10.0% w/v formulation was taken with stirring by a magnetic stirrer and diluted with 5% w/v gum arabic solution to prepare the 1.00 and 3.00% w/v formulations.
The formulations and vehicle were subdivided into plastic containers and stored at the cold place (target range: 1 to 10 °C). On each dosing day formulations and vehicle were taken out from the storage place and dosed to the animals. The formulations were used within a stable period of 16 days after preparation


VEHICLE
- Justification for use and choice of vehicle: The test material did not dissolve in purified water or olive oil, but was found to be homogenate with 5% w/v gum arabic solution at a concentration of 10% w/v (non-GLP). The vehicle was used for a general toxicity and historical control data are available.
- Concentration in vehicle: 1, 3 and 10 % w/v
- Amount of vehicle: 20 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the test item formulations at 10.0 and 1.00 w/v% were confirmed with high performance liquid chromatography (HPLC) as part of a different study (CERI study no. X02-0328) from the bottom, middle and upper part of the formulations. The coefficient of variations of the concentrations of each layer were found to be in an acceptable range and all formulations were confirmed to be homogenious. Dose formulations were confirmed to be stable after 16 days of storage in a cold place.

Concentrations of the test item formulations were confirmed with HPLC in the first preparation (CERI study no. X02-0328). Actual concentrations of 10.0, 3.00 and 1.00% w/v formulations were 10.2, 3.06 and 1.03% w/v, respectively and considered to be acceptable.

Duration of treatment / exposure:

Males were treated daily for 29 days, starting from 14 days prior to mating and including the mating period.
Females that delivered were treated daily for 14 days prior to mating, through mating and gestation until lactation Day 13.
Non-mating females of the satellite group were treated for 29 days.

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

Main group: 5 (control and high-dose group) and 10 (low- and mid-dose group)
Non-mating control: 10 (control and high-dose group only)
Recovery group: 10 (each 5 from the main group and 5 from the non-mating control; control and high-dose group only)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on the results of a 14-day repeated dose oral toxicity study (CERI study no. C21-0051). Groups of 3 rats/sex were administered dose levels of 50, 200, 500 and 1000 mg/kg bw/day. No clinical signs of toxicity were noted and all animals gained weight as expected. Necropsy and organ weights of the liver, kidneys and spleen revealed no abnormal findings. The top dose for the main study was therefore set to 1000 mg/kg bw/day.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for clinical signs and mortality during the treatment period and once daily during the recovery period. Females were observed for delivery and nursing conditions.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made prior to dosing and once weekly thereafter.
- Examinations included: Animal reactions for external stimuli, handling observations and arena observations.
Animal reactions for external stimuli: Holding animals or bringing hand close to hold, easiness of removal and vocalisation
Handling observations: Muscle tone, subnormal temperature, hair appearance including piloerection, stained hair and unkempt hair, skin and mucous colour such as paleness, reddening and cyanosis, eyes including lacrimation, exophthalmos and pupillary size, salivation and secretion
Arena observations: Posture, motor activity level, respiration, gait characteristics, lid closure, tremor, twitch, convulsion, stereotypical behaviour and abnormal behaviour for one min or more (within five min), frequencies of defecation (number of faeces) and urination (number of pools)

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded for males and non-mating females on Days 1, 3, 7, 14, 21 and 28 and on Days 1, 7 and 14 of recovery and at necropsy. For females, individual body weights were recorded on Days 1, 3, 7 and 14, on gestation Days 0, 7, 14 and 20 and on postnatal days 0, 4, 8 and 13. For non-copulated females, body weight was recorded on Days 21, 28, 35, 42 and 49.

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

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: Blood was collected at terminal sacrifice from the abdominal aorta.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/sex from the main group, 5 females from the non-mating group and males and females of the recovery group.
- Parameters examined: Red blood cell count (RBC), haemoglobin (Hb), haematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (Platelet), reticulocyte count ratio (Reticulo), white blood cell count (WBC), neutrophils (Neutro), lymphocytes (Lymph), eosinophils (Eosino), basophils (Baso) and monocytes (Mono), prothrombin time (PT) and activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected at terminal sacrifice from the abdominal aorta.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/sex from the main group, 5 females from the non-mating group and males and females of the recovery group.
- Parameters examined: Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (g-GTP), total cholesterol (T-Cho), triglycerides (TG), blood urea nitrogen (BUN), creatinine, total protein (T-Protein), albumin, A/G ratio, glucose, total bilirubin (T-Bil), total bile acids (TBA), inorganic phosphorus (IP), calcium (Ca), sodium (Na), potassium (K) and chloride (Cl)

PLASMA/SERUM HORMONES/LIPIDS: Yes
Blood was collected at terminal sacrifice from the abdominal aorta.
- Animals fasted: Yes
- How many animals: 5 males from the main group and 5 males from the recovery group.
- Parameters examined: Thyroid hormone (T4)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from each 5 animals/sex/group from the afternoon of the last observation day until the next morning, covering a period of 15-17 h.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Urine volume, colour, turbidity, specific gravity, pH, protein, glucose, occult blood and urinary sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In each last dosing week.
- Dose groups that were examined: 5 animals/sex from each group (main group, non-mating group and recovery group).
- Battery of functions tested: Sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All parental animals were subjected to a detailed gross necropsy including external surface of the body, all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities, and their contents after bleeding from the ventral aorta under isoflurane anesthesia on the next day of last dosing and last recovery days. Non-copulated female, non-delivered females and dead female were subjected as with a survived animal on each found day. Vaginal smears were collected before gross necropsy in survived females, and stages of the oestrous cycle were determined with a light microscope after Giemsa staining. The number of implantation sites of the uterus after incision were counted for the mated females. Organs and tissues were collected from all animals of all groups. Trachea, lungs and urinary bladder were inflated with 10% neutralized buffered formalin before removal. Stomach and intestines were filled and fixed with 10% neutralized buffered formalin and the contents were washed away with water.
The following organ weights were recorded: Liver, heart, kidneys, testes, epididymides, prostate (with a part of the urethra), seminal vesicles (with coagulating gland), ovaries, uterus, brain, spleen, thymus, thyroid (with parathyroid) and adrenals.


HISTOPATHOLOGY: Yes
All organs/tissues were preserved in 10% neutralized buffered formalin. Testes and epididymides were fixed in modified Davidson’s fixative. Decalcification was done for the bone and bone marrow (femur) with 10% formic acid formalin before trimming.
The following organs and tissues were examined:
Respiratory system: Trachea, lungs
Digestive system: Submandibular glands, stomach, intestines (duodenum to rectum, including Peyer's patches), pancreas, liver
Cardiovascular system: Heart
Urinary system: Kidneys, urinary bladder
Reproductive system: Testes, epididymides, prostate (ventral and dorsolateral lobes), seminal vesicles (including coagulating gland), ovaries, uterus (horn and cervix), vagina
Nervous system: Brain (including cerebrum, cerebellum and pons), spinal cord (thoracic) and sciatic nerve
Hematopoetic system: Bone marrow (femur), axillar lymph nodes, mesenteric lymph nodes, spleen and thymus
Endocrine system: Pituitary gland, thyroid (including parathyroid) and adrenals
Sense organ: Eyeballs
Musculoskeletal system: Skeletal muscle (femoral region), bone (femur)
Skin and acessory organ: Mammary gland

Light microscopic examinations were performed after embedding in paraffin, sectioning and hematoxylin and eosin (HE) staining in each 5 animals of the control and high dose groups. Spleen in females of the recovery group was examined since treatment-related changes were suspected in organ weight. All macroscopic lesions were examined.

Statistics:

Data on body weights of parental animals, food consumption, grip strength, locomotor activity count, haematology and blood chemistry, urine volume, urine specific gravity, organ weights, body weights on necropsy, mean oestrous cycle length, pairing days until copulation, gestation length, number of implantation sites, number of pups born, number of live pups, body weights of pups, AGD, number of nipples/areolae and T4 level of pups were analysed by Bartlett’s test for homogeneity of variance. If significant difference was not noted, the values of the control group and each test item group were analysed by Dunnet’s test. If significant difference was noted in the Bartlett test, the nonparametric Dunnet’s test was performed. The frequencies of defecation and urination were analysed by nonparametric Dunnett’s test. Body weights of pups were calculated on each sex as sample unit for each litter. Abnormal oestrous cyclicity, copulation index, conception index and delivery dam index were analysed by Fisher’s exact test between the control group and each test item group.
Indexes of delivery, birth, viability and sex ratio were examined by Bartlett’s test. If significant difference was not noted, the values of the control group and each test item group were analysed by Dunnet’s test. If significant difference was noted, the nonparametric Dunnet’s test was performed.
Data regarding body weights and food consumption during the recovery period, and parameters of haematology and blood chemistry, urine volume, urine specific gravity, organ weights and body weights at necropsy day for the recovery group were analysed by F-test for variance ratio. If there were no significant differences at a significance level of 5%, the Student t-test was performed. If there were significant differences at a significance level of 5% in the F-test, the Aspin-Welch test was performed. The frequencies of defecation and urination during the recovery period were analysed by Mann-Whitney U-test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, 1/10 females showed staining around the nose and mouth on the day of delivery. At 300 mg/kg bw/day, 1/10 animals showed incomplete treatment of placenta and died during delivery. The effects were not related to treatment. There were no findings in males.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no mortality at 1000 mg/kg bw/day. At 100 and 300 mg/kg bw/day, each 1/10 females died during delivery. The deaths were not related to treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant decrease in food consumption noted in males at 1000 mg/kg bw/day on Day 7 of the recovery period. As no findings were observed during the treatment-period and there were no related changes in body weight, the decrease was considered toxicologically not significant. For details, please refer to Table 1 under "Any other information on results incl. tables".

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):

In males at 100 mg/kg bw/day, MCHC was decreased (-2%). The finding was without any dose-response relationship and therefore not attributed to treatment.
There were no findings in mating females. In non-mating females, there was an increase in neutrophils (+60%) and a decrease in lymphocytes (-8%) at 1000 mg/kg bw/day. The findings were within the laboratory’s historical control range and, in the absence of any related changes, considered to be incidental.
After the recovery period, males of the 1000 mg/kg bw/day group showed an increase in reticulocytes (+20%), and females of the 1000 mg/kg bw/day group showed an elongated APTT (+1.5 s). The findings were not observed during the treatment period and/or within the laboratory’s historical control data and therefore not related to treatment.
For details, please refer to Table 2 under “Any other information on results incl. tables”.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant increase in chloride concentration in non-mating females at 1000 mg/kg bw/day (+2%). The finding fell within the laboratory’s historical control range and there were no related findings, therefore the change was considered to be incidental. For details, please refer to Table 2 under “Any other information on results incl. tables”.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: T4 hormone levels, epididymides weight and histopathology, oestrus cyclicity, liver weight, mammary gland histopathology, ovary weight and histopathology, prostate weight and histopathology, seminal vesicles (with coagulating glands) weight and histopathology, testes weight and histopathology, thyroid weight and histopathology, uterus (with cervix) weight and histopathology, vagina histopathology, vaginal smears, adrenals weight and histopathology, brain weight and pituitary histopathology, fertility, foetal development, gestation length, litter size, litter viability, litter/pup weight, number of implantations and corpora lutea, number of live births, pre- and post-implantation losses, presence of anomalies, reproduction and sex ratio.
For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, 1/10 males showed epithelial cells in the urine. In non-mating females, cloudy urine and white blood cells were observed at 0 and 1000 mg/kg bw/day without dose-relationship after treatment and after recovery. Epithelial cells were observed in the control group only. The findings were not related to treatment.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, females showed decreased motor activity in the 50-60 min interval. The change was considered as single occurrence since no abnormal changes were observed in total (0-60 min interval) and there were no related changes in other parameters. There were no findings at 100 or 300 mg/kg bw/day. For details, please refer to Table 3 under "Any other information on results incl. tables".

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In males, absolute (+17%) and relative (+23%) adrenal weight were increased at 100 mg/kg bw/day. In addition, relative thymus weight was decreased (-19%) at 100 mg/kg bw/day. In mated females, absolute adrenal weight was increased (+15%) at 300 mg/kg bw/day. In the absence of a dose-response relationship the findings were considered to be incidental.
After the recovery period, absolute (-1%) and relative (-10%) spleen weight were statistically significantly decreased in females at 1000 mg/kg bw/day. As no findings were noted during the treatment period and in the absence of histopathological findings, the effects were considered toxicologically not significant. For details, please refer to Table 4 under "Any other information on results incl. tables".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In females, macroscopic findings of spontaneous origin comprised blackish region and recessed region of the mucosa of the glandular stomach in all control and test item-treated groups. There was no dose-response relationship evident. For details, please refer to Table 5 under "Any other information on results incl. tables".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related findings. Any observations occurred sporadically in control and test item-treated animals. The dead female at 100 mg/kg bw/day had staining of the lower abdomen, dark reddish change and oedematous change of the lungs, perforation in the forestomach and blackish region of the mucosa of the glandular stomach. The dead female at 300 mg/kg bw/day showed staining in the lower abdomen, dark reddish change and oedematous change of the lungs, bilateral discoloration if the kidneys, small thymus, bilateral enlargement of the adrenals and hydrothorax.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone levels (T4) in males:
T4 levels were not affected by treatment at any dose level.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Summary of food consumption data in males (g/rat/day)

	Males
Dose (mg/kg bw/day)	0	100	300	1000
Group	Main + Recovery	Main	Main	Main + Recovery
Day 3	26.23 ± 1.68	26.80 ± 1.59	27.19 ± 1.89	27.18 ± 0.68
Day 7	26.31 ± 1.58	27.40 ± 2.27	27.39 ± 1.65	26.96 ± 1.46
Day 14	25.95 ± 1.28	26.65 ± 1.88	27.03 ± 1.82	26.23 ± 1.06
Day 28	26.16 ± 1.22	27.51 ± 1.50	27.08 ± 1.87	26.21 ± 1.77
Recovery period Day 7	26.62 ± 1.29	-	-	24.94 ± 0.90*
Recovery period Day 14	26.34 ± 2.16	-	-	26.20 ± 1.87
* significantly different from control at p < 0.05

Table 2: Summary of findings in haematology and clinical chemistry findings

Males

Females

Main group

Recovery group

Main group (Mating)

Non-mating

Recovery group (Mating)

Dose (mg/kg bw/day)

0

100

300

1000

0

1000

0

100

300

1000

0

1000

0

1000

Number of animals

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Haematology parameters

MCHC (g/dL)

36.44 ± 0.13

35.80 ± 0.51*

35.90 ± 0.38

36.32 ± 0.41

36.86 ± 0.51

36.52 ± 0.81

36.58 ± 0.54

36.60 ± 0.43

35.68 ± 0.78

36.18 ± 0.53

36.30 ± 0.46

36.34 ± 0.68

36.16 ± 0.49

36.18 ± 0.73

Reticulo (%)

4.034 ± 0.321

4.038 ± 0.533

3.888 ± 0.999

3.686 ± 0.375

3.302 ± 0.260

3.954 ± 0.354*

4.056 ± 1.321

3.286 ± 0.741

4.750 ± 2.303

4.258 ± 0.707

3.696 ± 0.550

3.756 ± 0.741

3.572 ± 0.371

3.980 ± 0.375

HCD mean Range: mean ± 2 SD

3.079 2.26 - 3.72 2.267 - 3.891

not reported

Neutro (%)

19.34 ± 7.26

17.10 ± 4.87

22.32 ± 5.41

18.52 ± 1.35

16.98 ± 1.19

21.18 ± 5.56

44.70 ± 6.44

45.32 ± 4.56

39.42 ± 7.82

37.48 ± 8.71

11.78 ± 4.45

18.84 ± 2.49*

18.48 ± 3.91

16.66 ± 6.48

HCD mean Range: mean ± 2 SD

not reported

20.49 11.4 - 36.2 3.95 - 37.03

Lymph (%)

74.72 ± 7.83

76.48 ± 5.26

71.70 ± 5.78

75.24 ± 1.20

75.60 ± 2.63

71.58 ± 6.79

48.84 ± 6.64

47.18 ± 4.83

53.92 ± 6.77

55.30 ± 7.25

83.22 ± 4.68

76.44 ± 2.77*

75.66 ± 4.90

78.00 ± 6.60

HCD mean Range: mean ± 2 SD

not reported

74.31 58.2 - 84.2 57.25 - 91.37

APTT (sec)

24.80 ± 2.63

25.10 ± 1.59

25.20 ± 1.62

24.48 ± 2.22

25.02 ± 1.19

24.86 ± 3.82

17.90 ± 0.57

18.44 ± 2.32

17.66 ± 1.61

17.24 ± 1.66

19.68 ± 1.29

18.14 ± 2.11

15.38 ± 2.09

19.40 ± 1.08**

HCD mean Range: mean ± 2 SD

not reported

16.63 9.4 - 18.9 12.37 - 20.89

Clinical chemistry parameters

Chloride (mEq/L)

106.22 ± 1.08

105.56 ± 0.83

106.10 ± 0.88

105.42 ± 0.46

105.18 ± 1.18

104.74 ± 1.13

100.60 ± 3.12

101.18 ± 2.14

102.28 ± 2.49

101.00 ± 2.37

106.42 ± 1.36

108.96 ± 1.00**

106.72 ± 1.25

105.84 ± 0.89

HCD mean Range: mean ± 2 SD

not reported

107.06 103.7 - 109.6 103.80 - 110.32

significantly different from control at * p < 0.05 and ** p < 0.01; MCHC: mean corpuscular haemoglobin concentration; Reticulo: reticulocytes; Neutro: neutrophils; Lymph: lymphocytes; APTT: activated partial thromboplastine time; HCD: historical control data

Table 3: Summary of findings on motor activity in males and non-mating females during Week 4 and in mating females during Week 7

	Males				Females
					Non-mating		Mating
Dose (mg/kg bw/day)	0	100	300	1000	0	1000	0	100	300	1000
Number of animals	5	5	5	5	5	5	5	5	5	5
0-10	45 ± 28	44 ± 36	30 ± 16	36 ± 6	74 ± 60	93 ± 21	48 ± 17	30 ± 21	30 ± 25	25 ± 31
10-20	19 ± 12	18 ± 11	16 ± 2	20 ± 10	29 ± 30	54 ± 30	36 ± 26	15 ± 13	9 ± 8	13 ± 15
20-30	8 ± 4	12 ± 14	18 ± 7	19 ± 10	25 ± 27	34 ± 24	13 ± 11	7 ± 11	20 ± 21	3 ± 4
30-40	7 ± 8	7 ± 6	5 ± 4	7 ± 10	8 ± 13	26 ± 21	22 ± 22	4 ± 5	7 ± 10	7 ± 11
40-50	9 ± 6	13 ± 13	6 ± 5	13 ± 14	3 ± 3	12 ± 18	3 ± 4	3 ± 5	5 ± 8	9 ± 18
50-60	2 ± 2	4 ± 6	7 ± 10	4 ± 3	7 ± 8	19 ± 15	32 ± 17	8 ± 11	13 ± 14	1 ± 1**
Total	91 ± 46	99 ± 63	83 ± 34	99 ± 36	145 ± 120	236 ± 67	153 ± 84	66 ± 23	84 ± 63	57 ± 53
**: significantly different from control at p < 0.01

Table 4: Organ weight findings

Males

Females

Main group

Recovery group

Main group (Mating)

Non-mating

Recovery group (Mating)

Dose (mg/kg bw/day)

0

100

300

1000

0

1000

0

100

300

1000

0

1000

0

1000

Number of animals

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Adrenals

Absolute weight (mg)

55.22 ± 3.53

69.81 ± 12.28*

64.75 ± 6.24

67.50 ± 12.82

62.02 ± 8.26

64.62 ± 7.09

73.21 ± 12.29

79.71 ± 10.86

84.44 ± 8.45*

74.57 ± 6.48

63.98 ± 13.00

70.92 ± 9.91

68.00 ± 7.07

68.68 ± 4.09

Relative weight (mg/100 g)

12.14 ± 1.15

14.97 ± 2.54*

13.87 ± 1.57

14.84 ± 2.36

12.58 ± 1.24

13.14 ± 1.75

24.59 ± 4.17

27.43 ± 3.17

28.27 ± 3.25

24.56 ± 2.07

24.78 ± 4.42

27.56 ± 3.33

24.32 ± 2.96

24.52 ± 2.84

Thymus

Absolute weight (mg)

504.76 ± 70.34

414.48 ± 75.77

450.27 ± 78.89

473.36 ± 41.05

387.86 ± 115.89

319.94 ± 82.94

198.35 ± 70.76

160.28 ± 36.57

182.62 ± 70.39

203.13 ± 39.22

411.42 ± 79.20

391.66 ± 98.01

348.94 ± 100.21

302.60 ± 78.16

Relative weight (mg/100 g)

110.26 ± 13.35

89.19 ± 17.03*

95.77 ± 12.61

104.34 ± 7.44

78.00 ± 18.93

65.00 ± 17.36

66.28 ± 21.98

55.40 ± 13.50

61.09 ± 23.42

67.06 ± 13.88

159.78 ± 28.74

151.66 ± 33.75

122.54 ± 24.70

106.24 ± 20.32

Spleen

Absolute weight (g)

0.690 ± 0.074

0.797 ± 0.106

0.812 ± 0.115

0.766 ± 0.186

0.762 ± 0.090

0.838 ± 0.124

0.498 ± 0.076

0.501 ± 0.049

0.531 ± 0.064

0.561 ± 0.083

0.516 ± 0.092

0.532 ± 0.071

0.612 ± 0.044

0.512 ± 0.081*

Relative weight (g/100 g)

0.152 ± 0.013

0.172 ± 0.022

0.172 ± 0.017

0.170 ± 0.042

0.156 ± 0.013

0.170 ± 0.030

0.166 ± 0.021

0.174 ± 0.018

0.179 ± 0.023

0.184 ± 0.026

0.200 ± 0.035

0.208 ± 0.033

0.220 ± 0.010

0.180 ± 0.025*

* significantly different from control at p < 0.05

Table 5: Gross necropsy findings in the glandular stomach

	Males						Females
	Main group				Recovery group		Main group (Mating)				Non-mating		Recovery group (Mating)
Dose (mg/kg bw/day)	0	100	300	1000	0	1000	0	100	300	1000	0	1000	0	1000
Number of animals examined	5	5	5	5	5	5	10	10	10	10	10	10	10	10
Glandular stomach
Blackish region of mucosa	0/5	0	0	0	0	0	5	4/0/1^#	4/0^$	3/0^§	1	0	0	0
Recessed region of mucosa	0	0	0	0	0	0	1	0/0/0^#	0/0^$	0/0^§	0	0	0	0
^#: scheduled sacrifice animal/ not copulated animal/ dead animal;^$: scheduled sacrifice animal/ dead animal;^§: scheduled sacrifice animal/ female animal did not deliver her pups

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, the NOAEL for systemic toxicity was 1000 mg/kg bw/day in male and female rats.