[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assesssment of the target substance

Adequacy of study:

key study

Justification for type of information:

refer to the analogue justification provided in IUCLID section 13

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: 302 - 380 g (males),180 - 247 g (females)
- Housing: In groups of three to five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK, batch/lot nos. 72 and 79 and ISO-BLOX from Harlan Laboratories B.V., Netherlands, batch/lot nos. T.6960C.CS-100099, 100267). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus
cycles.
- Diet (ad libitum): pelleted standard Harlan Teklad 2018C (batch nos. 43/12 and 56/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland)
- Water (ad libitum): community tap-water
- Acclimation period: at least seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): Based on an observation on 01-Feb-2013, the automatic light cycle of 12 h on / 12 h off was not functional within a block of rooms including room numbers 015A and 011 B. Due to the breeding performance from November 2012 onwards during this and other studies conducted in rooms which belong to the affected block, it was considered probable that the animals had been maintained under constant lighting throughout the study period for the first delivery animals. From 01-Feb-2013 onwards, the automatic light cycle was restored.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: daily. The test substance was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Amount of vehicle: 4 mL/kg bw (dose volume)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first dose formulation day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 1 g of each test item concentration were taken from the middle to confirm the stability (4 hours and 8 days at room temperature (20 ± 5°C)).
Towards the end of the study of the first and third delivery of animals, samples were taken from the middle to confirm concentration. Additional samples were taken on 21-Feb-2013 (first application of the second delivery of animals) from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice or at room temperature to Dr. G. Heinemann (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen /Switzerland) and stored there at -20 ± 5 °C until analysis or were analyzed directly.

As the results obtained were out of the accepted limits, a new analytical method was implemented, which necessitated additional dose formulation samples mainly from the low dose levels (for details please refer the analytical report). For the implementation of the method additional samples were taken during the study, which were not reported.

The samples were analyzed by derivatisation with MSTFA/BSTFA and analysis by GC-FID or analyzed by HPLC using a method which was implemented within the present study. Analyzed samples were not discarded without written consent from the study director.

Duration of treatment / exposure:

Males: Minimum 28 days
Females: Approximately 6 weeks

Frequency of treatment:

once daily (within four hours)

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

at least 12 (see any other information on materials and methods)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on single dose toxicity data of the test compound and repeated dose toxicity data of chemically similar compounds, the results of a 14-day range-finding study the dose levels of 100, 300, and 1000 mg/kg bw per day were selected for the present study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration of the test item and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum).
- detailed clinical observations were performed outside the home cage in a standard arena and the animals were observed for the following changes in: skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Yes
- Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly.
- Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 14 and 14 – 21 and days 1 - 4 of the lactation
No food consumption was recorded during the pairing period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day of the scheduled necropsy from 5 males randomly selected from each group. Blood samples from 5 lactating females from each group will be obtained on day 5 postpartum.
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes, for approximately 18 hours
- How many animals: 5 males randomly selected from each group and from 5 lactating females from each group
- Parameters checked: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular hemoglobin concentration, haemoglobin concentration distribution width, leukocyte count (total), differential leukocyte count: platelet count, reticulocytes; prothrombin time (= thromboplastin time), activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Animals fasted: Yes, for approximately 18 hours
- How many animals: see haematology
- Parameters checked:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Gamma-glutamyl-transferase
Bile acids
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males towards the end of the first pairing period and females on day 3 post partum
- Dose groups that were examined: five P generation males and five P generation females randomly selected from each group
- Battery of functions tested:
• Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
• Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
• Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
• Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
• Measurements / Counts: hind limb / fore limb grip strength, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately. In addition, from 5 males and 5 females killed at the end of the study which were selected for hematology and clinical chemistry examination from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
Adrenal glands (weighed as pairs)
Brain
Heart
Kidneys (weighed as pairs)
Uterus (including cervix)
Prostate
Liver
Thymus
Spleen
Thyroid (after fixation)
Ovaries (weighed as pairs)
Seminal vesicles (inclusive coagulating gland)

From all animals of the third delivery, following organs were weighed:
Testes
Epididymides
Heart
Liver
Kidneys (weighed as pairs)
Adrenal glands (weighed as pairs)

HISTOPATHOLOGY: Yes
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
Prostate
Seminal vesicles with coagulating gland
Testes (in Bouin’s fixative)
Epididymides (in Bouin’s fixative)
The following tissues from all parental females were preserved in neutral phosphate buffered 4%
formaldehyde solution:
Ovaries (with oviduct) Uterus (with vagina)

In addition, from all males and females the following tissues were preserved in neutral phosphate
buffered 4% formaldehyde solution:
Gross lesions
Brain
Spinal chord (cervical, thoracic, lumbar)
Small and large intestines (incl. Peyer’s patches)
Stomach (forestomach and glandular stomach)
Liver
Kidneys
Adrenals
Lymph nodes (axillary and mesenteric)
Urinary bladder
Aorta
Eyes with optic nerve and harderian gland
Lacrimal gland
Larynx
Nasal cavity
Esophagus
Heart
Thymus
Thyroids and parathyroids
Trachea and lungs (preserved by inflation
with fixative and then immersion)
Pituitary gland
Spleen
Peripheral nerve (sciatic)
Bone marrow (femur)
Femur with knee joint
Mammary gland (male and female)
Pancreas
Salivary glands – mandibular, sublingual
Skeletal muscle
Sternum with bone marrow
Pharynx

All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.
Testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of all three animal deliveries of the control and high-dose group were examined. The same applied to all occurring gross lesions. The remaining organs/tissues of 5 randomly selected males and females of the control and high-dose group, respectively, were examined histopathologically. From the 3th delivery of animals no additional organs/tissues of 5 randomly selected males and females of the control and high dose group were examined.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on the females that did not give birth. In addition, microscopic examination of the reproductive organs of males that did not successfully mate with the allocated female was performed.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables were 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 could not be assumed to follow a normal distribution.
• Fisher's exact-test will be applied if the variables could be dichotomized without loss of information

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Mortality:

mortality observed, treatment-related

Description (incidence):

see details on results

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see details on results

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):

see details on results

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

see details on results

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
At a dose level of 1000 mg/kg bw/day, one male (no. 171, third animal delivery) died spontaneously on day 1 of the after pairing period. No clinical signs were observed and a normal body weight gain was recorded until day 13 of the paring period. One day before he died he showed a body weight loss of 3%. During necropsy, a dark red discolored thymus was recorded.

BODY WEIGHT AND WEIGHT GAIN
Males:
Mean body weight gain was statistically significantly lower at 1000 mg/kg bw/day during the pre-pairing period, resulting in slightly reduced mean body weights (-3% at the end of this period compared to the controls). This was a result of the minor body weight loss (1%) between days 1 to 2. During the pairing period, body weight gain at the high dose level was similar to the control value. Due to this fact, the reduction in mean body weight gain and body weights were considered to be not adverse. The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and
1000 mg/kg bw/day were: +13%, +12%, +11% and +11% during the pre-pairing period, +5%, +5%, +3% and +4% during the pairing period and +7%, +9%, +7% and +8% during the after pairing period (percentages refer to the body weight gain within the period).

Females:
No effect of the test item on mean body weights and mean body weight gain was noted for females at any dose level. Towards the end of the gestation period, body weights and body weight gain were reduced in females at 1000 mg/kg bw/day (statistically significantly different body weight gains from day 17 post coitum onwards). These differences were considered to be a result of the increased post implantation loss and low number of pups, which were noted for dams at this dose level. The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +8%, +9%, +8% and +7% during the pre-pairing period, +63%, +57%, +57% and +51% during the gestation period and +4%, +7%, +4% and +3% during days 1 to 4 of the lactation period (percentages refer to the body weight gain within the period).

FOOD CONSUMPTION AND COMPOUND INTAKE
Males:
At 1000 mg/kg bw/day, mean food consumption was statistically significantly reduced during the first 11 days of treatment in males of the first and second delivery (-9.3% on day 11 of prepairing when compared with controls). Thereafter, the food consumption remained slightly lower than in controls, but without reaching statistical significance. Food consumption was also lower in males of the third delivery, but not to a statistically significant degree. Since this reduction in food consumption occurred only transiently, the reduction was considered to be not adverse.

Females:
There were no effects on mean food consumption at any dose level and in any study phase.

HAEMATOLOGY
The assessment of the hematology data did not reveal any test item-related effects in males and females at any dose level.

CLINICAL CHEMISTRY
The assessment of the clinical biochemistry data did not reveal any test item-related effects in males and females at any dose level.
Slightly higher urea levels were noted in males at 300 mg/kg bw/day and in females at 1000 mg/kg bw/day. Additionally, lower alkaline phosphatase levels were noted in females at 300 mg/kg bw/day, higher creatine levels in males and lower glucose levels in females at 1000 mg/kg bw/day, respectively. However, in the absence of dose-dependency and/or since values were additionally within the historical control range, a test item-related effect can be excluded.

NEUROBEHAVIOUR
No clinical signs were noted in males during the course of this study.
Incidentally, one control female (no. 45) showed a slightly decreased activity, ruffled fur and vaginal bleeding on day 25 post coitum. Necropsy of this female revealed masses in the uterus and vagina. Furthermore, one female (no. 66) treated at 100 mg/kg bw/day that did not give birth around day 21 post coitum either showed a wound on the anterior dorsum during the last three days prior to necropsy on day 25 post coitum. During necropsy reddish discolored ovaries and enlarged axillary lymph nodes were noted. Both females were pregnant.
These isolated findings were not related to treatment with the test item.

No findings were noted at detailed weekly clinical observation during the whole course of the
study.

There were no treatment-related changes noted at functional observational battery. One male at 100 mg/kg bw/day had increased faeces-balls and one female at 300 mg/kg bw/day showed decreased rearings. These isolated findings showing no dose relationship were deemed unrelated to treatment with the test item. A statistically significantly lower mean body temperature was noted in males of the first and second delivery at 1000 mg/kg bw/day (37.8 °C compared to 38.5 °C in the control group). No similar effect was noted for males treated at 100 and 300 mg/kg bw/day or in females. Since the value of 37.8°C was borderline to the range of the historical control data (38.0°C-38.6°C), and no change was noted in females, this finding was considered to be incidental.

ORGAN WEIGHTS
Treatment with the test item at 1000 mg/kg bw/day resulted in statistically significantly higher absolute and relative liver and kidney weights in animals of both genders, which was confirmed in animals of the third delivery except for kidney weights in females. Since there was no evidence for an impairment of organ function by clinical pathology and histopathology, these findings were not considered to be adverse. Additionally, in females, absolute and relative heart weights were also increased. This finding was without histopathological correlate, and was therefore considered to be incidental.
There were no effects of treatment at 100 and 300 mg/kg bw/day.

GROSS PATHOLOGY
All findings were typical of this strain and age of rat and were considered to be incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related findings.
In particular, qualitative examination of the stages of spermatogenesis in the testis did not reveal any treatment-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No treatment-related microscopic abnormalities were observed
in the evaluation of the ovarian follicles and corpora lutea of the ovaries or the evaluation of the uterus.
All findings were typical of this strain and age of rat and were 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: overall effects

Key result

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

Conclusions:

Based on these results, the NOAEL for general toxicity was considered to be 1000 mg/kg bw/day. The NOEL for general toxicity was established at 300 mg/kg bw/day.

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - 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:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: 352-426 g (males; mean: 372 g), 192-249 g (females; mean: 224 g)
- Housing: individual in stainless steel cages
- Diet: ad libtum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1 - 23.2
- Humidity (%): 48 - 61
- Air changes (per hr): more than 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was diluted in appropritate amounts of corn oil for each dose level. Aliquots of the dosing solution corresponding to the amount of daily administration were stored in the dark at 2 - 6 °C. The stability of the dosing solution was 7 days in a refrigerator and 1 day at room temperature. Therefore, the dosing solution was used within 7 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance showed low solubility in water.
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): V4N3566

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No details reported.

Duration of treatment / exposure:

males: 42 days (14 days prior to mating and 28 days thereafter)
females: 42-52 days (from 14 days before mating to day 4 of lactation)
satellite males and females: 42 days and 14 days post-exposure observation period

Frequency of treatment:

once daily, 7 days/week

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 females in control and test groups
7 males in control and 1000 mg/kg bw groups
12 males in 100 and 300 mg/kg bw groups
5 animals per sex in satellite control and 1000 mg/kg bw/day groups (in addition to number listed above)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Two dose range finding studies were performed. 2000 mg/kg bw of test substance was administered for 3 days in male and female rats. No abnormalities were found in general condition and body weight. The second study was performed at dose levels of 0, 330, 100, 300 and 1000 mg/kg bw/day for 14 days. No abnormalities of general condition, body weight, food consumption, hematological findings, blood biochemical findings, gross pathology and organ weight were found. Therefore, 1000 mg/kg bw/day was selected as the highest dose.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first exposure and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: Day 1 (before administration), 7, 14, 21, 28, 35 and 42 (before sacrifice)
Females: Day 1 (before administration), 7, 14, during pregnancy on Day 0, 7 14 and 21, during lactation on Days 0 and 4 (before sacrifice)
Satellite males and females: Day 1 (before administration), 7, 14, 21, 28, 35, 42, 49 (Day 7 of recovery period) and 56 (Day 14 of recovery period, before sacrifice)

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after last application
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters checked: RBC, Hemoglobin, Hematocrit, MCV, MCH, MCHC, Platelets, Reticulocytes, PT, APTT, WBC, Differential leukocytes: Lymphocytes, Neutrophils, Eosinophils, Basophils, Monocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after last application
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters checked: AST, ALT, ALP, γ-GTP, T-protein, Albumin, A/G, T-bilirubin, BUN, Creatinine, Glucose, T-cholesterol, Triglyceride, Na, K, Cl, Ca, P, LDH, choline esterase

URINALYSIS: Yes (males)
- Time schedule for collection of urine: Day 37 during administration period or Day 9 during recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Colour, cloudiness, water consumed, volume, specific gravity, Na, K, pH, Protein, Glucose, Ketone body, Bilirubin, Occult blood, Urobilinogen, Epithelial cells, Erythrocytes, Leukocytes, Casts, Crystals, Fat

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Males: on Week 6 of the administration period
Females: on Week 6 of the administration period
Males and females of satellite groups: on Week 6 of the administration period and on Week 2 of the recovery period
- Dose groups that were examined: all
- Battery of functions tested: hearing reaction, eye sight reaction, sense of touch reaction, pain reaction, pupil reflex, pinna reflex, ipsilateral flexor reaction, eyelid reflex, righting reflex

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Body surface, mucous membranes and internal organs

HISTOPATHOLOGY: Yes
Brain, pituitary, thyroid, thymus, lung trachea (after liquid immersion fixation), stomach, intestines, heart, liver, spleen, kidney, adrenal gland, bladder, testis, epididymis, prostate, seminal vesicles, ovaries, uterus, spinal cord (cervical, thoracic, lumbar), sciatic nerve, bone marrow (femur), lymph nodes (cervical lymph node, mesenteric lymph nodes), mammary gland, and other gross abnormalities
Spermatogenic cycle (Stage II, III, V, VII, and XII) was also investigated.

SACRIFICE
- Males: on Day 43
- Females: on Day 5 of lactation
- Females (unsuccessful mating): on Day 53
- Females (mated but non-pregnant): on Day 5 after scheduled delivery
- Satellite males and females: on Day 15 of the recovery period

Other examinations:

Organ weight: brain, liver, kidney, spleen, heart, thymus, thyroid, pituitary, adrenal, testis, sminal vesicle, epididymis
Estrous cycle
Number of ovarian corpora lutea
Number of uterine implantation
Observation of pups

Statistics:

ANOVA, Barlettm Kruskal-Willis, Dunnett, F test, Studen t-test, Aspin-Welch t-test, Mann-Whitney U-test, Fisher's exact test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day (satellite group, males): significant increase was observed (non adverse)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day (satellite group, females): significant increase on Day 14 of administration (non adverse)

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):

100 and 300 mg/kg bw/day (males): decrease in APTT without dose-dependency after administration period (non adverse)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

300 and 1000 mg/kg bw/day (females): significant increase in organic P in females after administration period without dose-dependency (non adverse); 1000 mg/kg bw/day (satellite group, females): significant increase in choline esterase (non adverse)

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: significant decrease in absolute weight of seminal vesicles in male and significant decrease in relative weight of spleen in females after administration period (non adverse)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/day: mass in subcutis in one female after administration period (not treatment-related)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

control and 1000 mg/kg bw/day: after administration period, low incidence of commonly found microscopic changes, evenly distributed between groups (not treatment-related)

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/day: after administration period, benign fibroadenoma of the mammary gland in one female (not treatment-related)

Details on results:

CLINICAL SIGNS AND MORTALITY
No abnormalities and no mortality were observed.

BODY WEIGHT AND WEIGHT GAIN
No change of body weight and weight gain was observed during the administration period. During the recovery period, a significant increase in body weight was noted in males at 1000 mg/kg bw/day. This was caused by a tendency of the control group animals to lose weight. One male in control group showed a significant decrease in body weight during the recovery period. However, no other abnormalities were observed in this male.

FOOD CONSUMPTION
No change was observed during the administration period in the test groups. A significant increase in food consumption was found in satellite females of the 1000 mg/kg bw/day group on Day 14 of administration. However, it was regarded as an incidental finding since the food consumption of the corresponding control group was relatively small on that day. Therefore, this change was not regarded as a compound-related effect.

HAEMATOLOGY
No changes were observed. After administration, a significant decrease in APTT was observed in males of the 100 and 300 mg/kg bw/day groups in comparison with control group. However, this change was not regarded as compound-related, because no dose-dependency was observed and this was within reference range.

CLINICAL CHEMISTRY
A significant decrease in inorganic P was observed in females of the 300 and 1000 mg/kg bw/day groups after administration. However, this change was not regarded as compound-related, since a dose-dependency was not observed and almost all the individual data were within the reference ranges except for one female of 300 mg/kg bw/day.
Choline esterase was increased in females of 1000 mg/kg bw/day after the recovery period but this was within the reference range. This change was not compound-related effect since this change was slightly and no change was observed after the administration period.

URINALYSIS
No significant changes were found during the administration and recovery periods.

NEUROBEHAVIOUR
No abnormality was observed for the sensory/reflex function, landing foot, grip strength and motor activity during the administration period. During the recovery period, motor activity increased from 0 to 60 min but not from 0 to 30 min in females dosed 1000 mg/kg bw/day. This effect was not compound-related since the observed change was slight and no change was observed during and after the administration period.

ORGAN WEIGHTS
After the administration period, a significant decrease in the absolute weight of seminal vesicles in males given and a significant decrease in relative weight of spleen in females were observed in the 100 mg/kg bw/day group. However, this change was not compound-related because no abnormalities were found at histopathological examination and there was no dose-dependency.
After the recovery period, a significant decrease in relative weight of pituitary in males of the 1000 mg/kg bw/day group and relative weight of thyroid in females of the 1000 mg/kg group bw/day was observed. Nevertheless, this effect was not compound-related because no abnormality was reported regarding these organs after administration period.

GROSS PATHOLOGY
No test substance-related changes were found. No abnormalities were found in breeding pairs which were not successful at mating and in those which were successful at mating but not pregnant.
After administration period, reddish thymus was noted in one male of the control group and subcutis mass was found in one female of 300 mg/kg by/day group (see also HISTOPATHOLOGY: NEOPLASTIC). After the recovery period, larger spleen and capsular thickening in spleen was found in one male and reddish area in thymus was observed in one female at 1000 mg/kg bw/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test substance-related changes were observed. There were also no changes regarding spermatogenic cycle.
Myocardial degeneration/fibrosis, foam cell accumulation in lung, mineralization in artery in lung, fatty degeneration of hepatocyte, microgranuloma in liver, solitary cyst in kidney, hyaline cast in kidney, lymphocyte infiltration in cortex of kidney, mineralization of cortico-medullary junction in kidney, fibrosis of cortex in kidney and haemorrhage in thymus were observed both in the control and 1000 mg/kg bw/day groups or only in the control group with low incidence. Hyaline droplet of proximal tubular epithelium in kidney was found in all males of the control and 1000 mg/kg bw/day groups. Brown deposit pigment and extramedullary haematopoiesis in spleen were found in all males and females of the control and 1000 mg/kg bw/day groups. However, there were no differences between control and test group. In the 1000 mg/kg bw/group, lymphocyte interstitium infiltration in prostate was found in one male, interstitial focal inflammation in lung and focal necrosis in liver was observed in one female. These changes occur naturally and were not compound-related.
No abnormalities were found in uterus and ovary in the non-pregnant females and the unsuccessful copulation females of the control and 1000 mg/kg bw/day groups, respectively. Degeneration of seminiferous tubules of testis, decrease in sperm and atrophy in prostate was observed in one control male.

HISTOPATHOLOGY: NEOPLASTIC
Mass on abdomen was found in a female after 40 days of administration in the 300 mg/kg bw/day group. However, this subcutaneous tumour of the mammary gland was a benign fibroadenoma and generated naturally.

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: overall effects

Critical effects observed:

not specified

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered), esters with glycerol oligomers

IUPAC Name:

Fatty acids, C16-18 (even numbered), esters with glycerol oligomers

Results and discussion

Results of examinations

Description (incidence and severity):

see details on results

Effect levels

1

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: overall effects

Remarks on result:

other:

Remarks:

Source CAS 63705-03-3

2

Key result

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Remarks on result:

other:

Remarks:

Source CAS 63705-03-3

3

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: overall effects

Remarks on result:

other:

Remarks:

Source CAS 111-03-5

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

As detailed in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in repeated dose systemic toxicity potential. With both source substances, the NOAEL for general systemic toxicity was determined to be 1000 mg/kg bw/day. Therefore, the NOAEL for general systemic toxicity for the target substance is also expected to be 1000 mg/kg body weight/day.