Registration Dossier

Administrative data

Description of key information

Three well conducted oral studies up to 90 day duration were conducted with the registration substance. The NOAEL was the limit dose of 1000 mg/kg/day in all three studies.

The most appropriate route of exposure for hazard evaluation is the oral route as this is where the systemic dose will be highest. Exposure via inhalation is not considered to be of importance because the substance is non-volatile. Furthermore, the substance is used under industrial conditions where correct training and strict adherence to PPE and general risk management measures is expected. Additional testing is therefore contraindicated on the grounds of animal welfare and lack of probable exposure via the inhalation route.

The test substance is of relatively large molecular weight and it's PChem properties (high Log Kow) dictate that absorption through unbroken skin will be negligible. Furthermore, the substance is used under industrial and professional conditions where correct training and strict adherence to PPE and risk management measures are expected. The most appropriate route of exposure for hazard evaluation is therefore likely to be the oral route, as this will result in the greatest systemic dose.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
October 2012 - November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study fully reported. No formal guideline as this study is a range-finding study.
Qualifier:
no guideline available
Principles of method if other than guideline:
The objective of this study was to evaluate the potential toxicity of EXP1200078 when administered daily by oral gavage to Sprague Dawley rats for 14 consecutive days and to assist in the dose selection for subchronic study(ies).
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): EXP1200078
- Physical state: dark brown viscous liquid
- Storage condition of test material: at room temperature in the dark

- Analytical purity: 100%
- Lot/batch No.: E00275-350
- Expiration date of the lot/batch: 31 Dec 2013
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: approximately 38 days old at receipt
- Housing: clean, stainless steel, wire mesh cages suspended above cage board
- Diet: ad libitum PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal)
- Water: ad libitum
- Acclimation period: 13 day acclimation/pretest period.

ENVIRONMENTAL CONDITIONS
- Temperature: 20.8°C to 21.8°C
- Humidity: 44.1% to 48.1%
- Air changes: 10 fresh air changes per hour

Each group (Groups 1-4) consisted of 5 males and 5 females. The animals were approximately 7 weeks old at the initiation of dose administration. Individual body weights ranged from 193 g to 233 g for males and from 158 g to 191 g for females at randomization.
- Photoperiod: 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod.

IN-LIFE DATES: From: 31 October 2012 to: 14 November 2012
Route of administration:
oral: gavage
Vehicle:
other: mineral oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18°C to 24°C). The test substance formulations were stirred continuously throughout the preparation and dose administration procedures.

VEHICLE
The vehicle used in preparation of the test substance formulations and for administration to the control group was mineral oil (lot no. 1AL0557, exp. date: 31 October 2014, manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ).

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The formulations were not analyzed for this non-GLP study.
Duration of treatment / exposure:
The vehicle and test substance formulations were administered orally by gavage via an appropriately sized flexible Teflon® shafted, stainless steel ball tipped dosing cannula once daily for 14 consecutive days, through the day prior to the scheduled necropsy. The dose volume for all groups was 5 mL/kg.
Frequency of treatment:
Daily.
Remarks:
Doses / Concentrations:
0, 100, 300 or 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Each group (Groups 1-4) consisted of 5 males and 5 females.
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: There were no signs of toxicity at up to 2000 mg/kg in an acute oral toxicity study (Hurley, Draft, WIL 537027). Therefore, in this 14-day study, the high-dosage level was the limit dose of 1000 mg/kg/day and the low- and mid-dose dosage levels provided an appropriate range to assist in the dose selection for subsequent subchronic study(ies).
Positive control:
No positive control included.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
Clinical examinations were performed twice daily, at the time of dose administration and approximately 1 to 2 hours following dose administration. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Detailed physical examinations were conducted on all animals during the pretest period within 2 days of receipt, one week (± 2 days) prior to randomization, on the day of randomization, weekly (± 2 days) during the study period, and on the day of the scheduled necropsy.

BODY WEIGHT: Yes
Individual body weights were recorded during the pretest period within 2 days of receipt, one week (± 2 days) prior to randomization, on the day of randomization, on study day 0 (prior to dosing), weekly (± 2 days) during the study period, and on the day prior to the scheduled necropsy. Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION: Yes
Individual food consumption was recorded one week (± 2 days) prior to randomization, on the day of randomization, and weekly (± 2 days) during the study period. Food intake was calculated as g/animal/day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Clinical findings that were confirmed macroscopically were designated CEO on the individual macroscopic data tables. The following tissues and organs were collected and placed in 10% neutral buffered formalin (except as noted) for possible further microscopic analysis:

Adrenals (2)
Aorta
Bone with marrow: Femur and Sternum
Bone marrow smear (from femur)
Brain
Cervix
Epididymides (2)
Eyes with optic nerve (2)
Gastrointestinal tract: Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum
Heart
Kidneys (2)
Larynx
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by inflation with fixative)
Lymph nodes: Axillary (2), Mandibular (2), Mesenteric
Ovaries with oviducts (2)
Pancreas
Peripheral nerve (sciatic)
Peyer’s patches
Pharynx
Pituitary
Prostate
Salivary glands (mandibular [2])
Seminal vesicles (2)
Skeletal muscle (rectus femoris)
Skin (with mammary gland)
Spinal cord (cervical, thoracic, lumbar)
Spleen
Testes (2)
Thymus
Thyroid (with parathyroids, if present [2])
Tongue
Trachea
Urinary bladder
Uterus
Vagina
Gross lesions (when possible)
Other examinations:
The following organs were weighed from all animals at the scheduled necropsy:
Adrenals
Brain
Epididymides
Kidneys
Liver
Ovaries with oviducts Pituitary
Spleen
Testes
Thymus
Thyroid with parathyroids*
Uterus

Paired organs were weighed together. Designated organs (*) were weighed after fixation. Organ to final body weight and organ to brain weight ratios were calculated.
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables.
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Body weight, body weight change, food consumption, and organ weight data were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance treated groups to the control group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals survived to the scheduled necropsy. Test substance-related clinical observations of clear material around the mouth were noted in the 1000 mg/kg/day group males. These observations were noted with low incidence (only once on study days 6, 7, 9, and 13) at approximately 1-2 hours following dose administration in 3 of 5 males in the 1000 mg/kg/day group. All other clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose related manner, and/or were common findings for laboratory rats of this age and strain.
Mortality:
mortality observed, treatment-related
Description (incidence):
All animals survived to the scheduled necropsy. Test substance-related clinical observations of clear material around the mouth were noted in the 1000 mg/kg/day group males. These observations were noted with low incidence (only once on study days 6, 7, 9, and 13) at approximately 1-2 hours following dose administration in 3 of 5 males in the 1000 mg/kg/day group. All other clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose related manner, and/or were common findings for laboratory rats of this age and strain.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights were unaffected by test substance administration. There were no statistically significant differences when the control and test substance treated groups were compared.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by test substance administration.
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
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights were unaffected by test substance administration.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test substance related macroscopic findings at the scheduled necropsy.
Histopathological findings: non-neoplastic:
not examined
Dose descriptor:
dose level: 1000 mg/kg bw/day
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Critical effects observed:
not specified
Conclusions:
All animals survived to the scheduled necropsy. There were no test substance-related effects on body weight, food consumption, or organ weights. There were no test substance-related gross observations noted. Test substance-related clinical observations of clear material around the mouth were noted with low incidence in the 1000 mg/kg/day group males.


Based on the results of this study, oral administration of EXP1200078 to Crl:CD(SD) rats for 14 consecutive days resulted in a maximum tolerated dose (MTD) of 1000 mg/kg/day. At this latter dose level, test substance-related effects were limited to low incidence of clear material around the mouth noted in males.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2012-February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
EXP1200078
Lot no. E00275-350
Exp. date: End-2013
Dark brown, opaque, viscous liquid
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 51 days
- Weight at study initiation: male 147-168 grams, female 127-152 grams
- Fasting period before study: prior to clinical pathology blood collection
- Housing: Upon arrival animals were housed 2 to 3 per cage by sex for approximately 3 days. Thereafter, all animals were housed individually in stainless steel, wire-mesh cages.
- Diet: ad libitum PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal)
- Water: ad lbitum Reverse osmosis-treated (on-site) drinking water
- Acclimation period: 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-22.1
- Humidity (%): 32.9-54.9
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 December 2012 To: 02 January 2013
Route of administration:
oral: gavage
Vehicle:
other: mineral oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle suspension was dispensed approximately weekly for administration to the control group and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature. The vehicle was mixed throughout the dispensation, sampling, and dose administration procedures.The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: (0, 20, 60 and 200 mg/mL)
- Amount of vehicle (if gavage): 5 mL/kg
- Lot no.: 1AL0557
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test substance formulations at concentrations that bracket those used on the present study was confirmed in a previous study (Coffee, 2013, WIL 537029) following 5- and 12-day storage at room temperature. Therefore, additional assessments of formulation stability were not conducted.
Quadruplicate samples for homogeneity determination were collected from the top, middle, and bottom strata of the first preparation of the 20 and 200 mg/mL dosing formulations. The last aliquot used for dosing from the first formulation preparation at these concentrations was also sampled for resuspension homogeneity (quadruplicate samples collected from the top and bottom strata). Quadruplicate samples for concentration analysis were collected from the middle stratum of the dosing formulations prepared for use during study weeks 0 and 3 (including the control group). One duplicate set was analyzed and the remaining duplicate set was stored at room temperature and retained as backup samples. All analyses were conducted by the WIL Research using a validated high performance liquid chromatography method using fluorescence detection.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Mineral oil vehicle
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:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 14-day oral range-finding toxicity study (Haas, 2013, WIL-537025) in which there were non-adverse findings of wet clear material around the mouth at 1 to 2 hours post-dosing in the 1000 mg/kg/day group males.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: within 3 days of receipt, 1 week (± 2 days) prior to randomization, on the day of randomization, weekly (± 2 days) during the study, and on the day of the scheduled necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: within 3 days of receipt, 1 week prior to randomization, on the day of randomization, prior to dosing on study day 0, weekly during the study, and on the day prior to the scheduled necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/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 AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study day 28
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters according to guideline were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study day 28
- Animals fasted: Yes
- How many animals: all animals
- Parameters according to guideline were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: study day 28
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters according to guideline were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during pretest and during study week 3 (prior to dose administration)
- Dose groups that were examined: all groups
- Battery of functions tested: FOB: sensory activity / grip strength / motor activity / handling observations / open field observations / neuromuscular observations / physiological observations

OTHER: organ weight
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. A complete necropsy was conducted on all animals. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The following tissues and organs were collected and placed in 10% neutral-buffered formalin:
Adrenals (2)
Aorta
Bone with marrow
Femur
Sternum
Bone marrow smear (from femur) a
Brain
Cervix
Epididymides (2) b
Eyes with optic nerve (2) c
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys (2)
Larynx
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by inflation with fixative)
Lymph nodes
Axillary (2)
Mandibular (2)
Mesenteric Ovaries with oviducts (2) d
Pancreas
Peripheral nerve (sciatic)
Peyer’s patches
Pharynx
Pituitary
Prostate
Salivary glands (mandibular [2])
Seminal vesicles (2)
Skeletal muscle (rectus femoris)
Skin (with mammary gland) e
Spinal cord (cervical, lumbar, thoracic)
Spleen
Testes (2) b
Thymus
Thyroid (with parathyroids, if
present [2]) d
Tongue
Trachea
Urinary bladder
Uterus
Vagina
Gross lesions (when possible)

a = Bone marrow smears were obtained at scheduled necropsy, but not placed in formalin; slides were not examined.
b = Fixed in modified Davidson’s solution.
c = Fixed in Davidson’s solution.
d = Parathyroids and oviducts were examined if in the plane of section and in all cases where a gross lesion of the organ was present.
e = For females; a corresponding section of skin was taken from the same anatomic area for males.


HISTOPATHOLOGY: Yes. After fixation, protocol-specified tissues were trimmed and processed into paraffin blocks, sectioned at 4 to 5 microns, mounted on glass microscope slides, and stained with hematoxylin and eosin.
Microscopic examination was performed on all tissues mentioned by gross pathology from the animal found dead and from all animals in the control and 1000 mg/kg bw/day groups at the scheduled necropsy. In addition, gross lesions were examined microscopically from all animals in the 100 and 300 mg/kg bw/day groups.
Statistics:
Conducted by WIL Research: All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Body weight, body weight change, food consumption, continuous FOB, clinical pathology, and organ weight data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. FOB parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test (Steel and Torrie, 1980).
Conducted by BioSTAT Consultants, Inc: All statistical analyses were conducted using SAS® version 9.2 (SAS Institute, Inc., 2002-2008), or higher. The SAS® procedure PROC MIXED was used for analysis with the random effect of animal included as the repeated measurement. The covariance structure across time was selected by comparing Akaike’s Information Criterion (AIC).
The monotonic dose-response relationship was evaluated using sequential linear trend tests based on ordinal spacing of dose levels. The linear dose by time interaction (LinTrt*Time) was evaluated. If the linear dose by time interaction was not significant, the trend test was conducted across the pooled time intervals for the entire session only.
Nonmonotonic dose responses were evaluated whenever no significant linear trends were detected but TRT and/or TRT*TIME interaction was significant at the 0.01 level. Within the framework of the RANOVA, pairwise comparisons were made for each individual test substance-treated group with the control group through linear contrasts. These nonmonotonic dose-response comparisons were conducted at the 0.01 significance level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
test substance-related clear material around the mouth
Mortality:
mortality observed, treatment-related
Description (incidence):
test substance-related clear material around the mouth
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
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):
Test substance-related higher mean liver weights were noted in the 1000 mg/kg/day group males and females and a lower mean seminal vesicles weight was noted in the 1000 mg/kg/day group males.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no test substance-related deaths. At a low incidence, test substance-related clear material around the mouth was noted in the 1000 mg/kg/day group males (only noted 1 to 2 hours after dose administration beginning on study day 11) and females (at the time of dosing and 1 to 2 hours post-dosing beginning on study day 8).

HAEMATOLOGY: Lower mean absolute neutrophil counts were noted in the 100 and 300 mg/kg/day group males; however, these changes did not show a clear dose-related response. Slightly higher, not statistically significant, mean white blood cell counts and mean absolute lymphocyte counts were noted in the 1000 mg/kg/day group males and females. However, the group means and all individual animal values for the 1000 mg/kg/day group males and females were within the WIL Research historical control database ranges. These observed changes were due to individual animal biological variation and were not considered to be test substance related.

ORGAN WEIGHTS: Higher mean liver weights (absolute, relative to final body weight, relative to brain weight) were noted in the 1000 mg/kg/day group males (12.6%, 10.5%, and 14.8%, respectively) and females (11.5%, 7.0%, and 10.3%, respectively); differences were not statistically significant in the females. However, all individual animal absolute liver weight values were within the WIL Research historical control database range of ± 2 standard deviations from the mean with the exception of 1 individual animal value in the 1000 mg/kg/day group females. There were no clinical pathology and/or histologic changes correlating to the higher liver weights.
A lower mean seminal vesicles weight relative to final body weight (17.7%) was noted in the 1000 mg/kg/day group males. However, the lower mean absolute seminal vesicles weight and mean seminal vesicles weight relative to brain weight differences were not statistically significant (16.2% and 14.9%, respectively), all individual animal absolute seminal vesicle weight values were within the WIL Research historical control database reference range, and there were no histologic changes correlating to the lower seminal vesicles weight. This change was considered to be toxicologically unimportant.


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: see 'Remark'
Critical effects observed:
not specified

The analyzed dosing formulations were within WIL Research’s SOP range for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the vehicle formulation that was administered to the control group.

Executive summary:

EXP1200078 was administered by daily oral gavage during 28 days to Crl:CD rats (5/sex) at dose levels of 0, 100, 300 and 1000 mg/kg/day, according to OECD guideline 407.

There were no test substance-related deaths. Body weight, food consumption, functional observational battery, locomotor activity, hematology, coagulation, serum chemistry, and urinalysis parameters were unaffected by test substance administration. There were no test substance-related gross necropsy observations or histologic changes.

Test substance-related clinical observations of clear material around the mouth were noted in the 1000 mg/kg/day group males and females.

Test substance-related higher liver weights were noted in the 1000 mg/kg/day group males and females and a lower seminal vesicles weight was noted in the 1000 mg/kg/day group males. These observations were not accompanied by histopathological changes, and considered to be not relevant/adverse.

Based on the results of this study, the NOAEL was considered to be 1000 mg/kg/day, the highest dosage level administered.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2012 - June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: In accordance with OECD guideline and GLP
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
EXP1200078
Batch/Lot no. E00048-256
Exp. date: End-2014
Dark brown, opaque, viscous liquid
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratores, Inc., Raleigh, NC
- Age at study initiation: animals were approximately 7 weeks old at the initiation of dose administration
- Weight at study initiation: individual body weights ranged from 196 g to 237 g for males and from 156 g to 195 g for females at randomization
- Fasting period before study: no
- Housing: individually in clean, stainless steel, wire mesh cages suspended above cage board
- Diet: ad libitum, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal)
- Water: ad libitu, reverse osmosis treated (on site) drinking water, delivered by an automatic watering system
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6°C to 22.2°C
- Humidity (%): 37.4% to 67.7%
- Air changes (per hr): 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod

IN-LIFE DATES: From: 22 January 2013 to: 23 April 2013
Route of administration:
oral: gavage
Vehicle:
other: Mineral oil, USP (lot nos. 1AL0557, 2BK0397, and 2BC0539, retest dates: 31 October 2014, 31 October 2014, and 28 January 2015, respectively, manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ)
Details on oral exposure:
The vehicle and test substance formulations were administered orally by gavage via an appropriately sized flexible Teflon® shafted, stainless steel ball tipped dosing cannul
PREPARATION OF DOSING SOLUTIONS:
The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature (18°C to 24°C). The vehicle was mixed throughout the dispensation, sampling, and dose administration procedures.

VEHICLE
The vehicle used in preparation of the test substance formulations and for administration to the control group was mineral oil, USP (lot nos. 1AL0557, 2BK0397, and 2BC0539, retest dates: 31 October 2014, 31 October 2014, and 28 January 2015, respectively, manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ).

Adjusted doses became effective the day of collection of the weekly body weights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test substance formulations at concentrations that bracket those used on the present study was confirmed in a previous study (Coffee, 2013, WIL-537029) following 5- and 12-days of storage at room temperature.
Homogenity determination sampels were collected from the top, middle, and bottom strata fo th e first preparation of the 20 and 200 mg/ml dosing formulations.
Samples for concentration analysis were collected from the middle stratum of the dosing formulations prepared for use during study weeks 0, 4, 8 and 12.
The analyzed dosing formulations were found to contain 94.0% to 104% of the test substance which was within WIL Research’s SOP range of target concentrations for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the vehicle formulation that was administered to the control group (Group 1).
Duration of treatment / exposure:
The vehicle and test substance formulations were administered orally by gavage via an appropriately sized flexible Teflon® shafted, stainless steel ball tipped dosing cannula once daily for 90 or 91 consecutive days, through the day prior to the scheduled necropsy. The dose volume for all groups was 5 mL/kg.
Frequency of treatment:
Daily.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Mineral oil control; vehicle for dosing.
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:
Each group consisted of 10 males and 10 females.
Control animals:
yes, concurrent vehicle
Details on study design:
Dosage levels were determined from the results of a 28-day oral toxicity study (Haas, 2013, WIL-537026) in which there were no dose-limiting findings at dosages up to 1000 mg/kg bw/day.
Positive control:
No positive control included, not required.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
Clinical examinations were twice daily, at the time of dose administration and 1 to 2 hours following dose administration. The absence or presence of findings was recorded for individual animals at the scheduled intervals.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed physical examinations were conducted on all animals 1 week (± 2 days) prior to randomization, on the day of randomization, weekly (± 2 days) during the study period, and on the day of the scheduled necropsy.

BODY WEIGHT: Yes
Individual body weights were recorded 1 week (± 2 days) prior to randomization, on the day of randomization, prior to dosing on study day 0, weekly (± 2 days) during the study period, and on the day prior to the first day of the scheduled necropsy (nonfasted). Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION: Yes
Individual food weights were recorded 1 week (± 2 days) prior to randomization, on the day of randomization, and weekly (± 2 days) during the study period. Food consumption was calculated as g/animal/day for the corresponding body weight intervals.

OPHTHALMOSCOPIC EXAMINATION: Yes
Ocular examinations were conducted on all animals prior to randomization and during the last week of the dosing period. All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the scheduled necropsy (study week 12/13)
- Anaesthetic used for blood collection: Yes, blood was collected for hematology and serum chemistry evaluation via the retro-orbital sinus of animals anesthetized by inhalation of isoflurance. Blood was collected for coagulation parameters at the time of euthanasia via the vena cava of animals euthanized by inhalation of carbon dioxide.
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined:
Total leukocyte count (WBC)
Erythrocyte count (RBC)
Hemoglobin (HGB)
Hematocrit (HCT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet count (PLATELET)
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)
Reticulocyte count, Percent (RETIC) and Absolute (RETIC ABSOLUTE)
Mean platelet volume (MPV)
Differential leukocyte count, percent and absolute:
-Neutrophil (NEU)
-Lymphocyte (LYMPH)
-Monocyte (MONO)
-Eosinophil (EOS)
-Basophil (BASO)
-Large unstained cell (LUC)
Red cell distribution width (RDW)
Hemoglobin distribution width (HDW)
Platelet estimate
Red cell morphology (RBC Morphology)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the scheduled necropsy (study week 12/13)
- Animals fasted: Yes
- How many animals: all
- Parameters examined:
Albumin
Total protein
Globulin [by calculation]
Albumin/globulin ratio (A/G Ratio) [by calculation]
Total bilirubin (Total Bili)
Urea nitrogen
Creatinine
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma glutamyltransferase (GGT)
Glucose
Total cholesterol (Cholesterol)
Calcium
Chloride
Phosphorus
Potassium
Sodium
Sorbitol dehydrogenase (SDH)
Triglycerides (Triglyceride)
Appearance

URINALYSIS: Yes
- Time schedule for collection of urine: at the scheduled necropsy (study week 12/13)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:
Specific gravity (SG)
pH
Urobilinogen (URO)
Total volume (TVOL)
Color (COL)
Clarity (CLA)
Protein (PRO)
Glucose (GLU)
Ketones (KET)
Bilirubin (BIL)
Occult blood (BLD)
Leukocytes (LEU)
Nitrites (NIT)
Microscopy of sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during study week 11/12 (prior to dose administration)
- Dose groups that were examined: all
- Battery of functions tested: Home cage observations, handling observations, open field observations, sensory observations, neuromuscular observations, physiological observations and locomotor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

ORGAN WEIGHTS: Yes
The following organs were weighed from all animals at the scheduled necropsy:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries with oviducts
Pituitary
Prostate
Seminal vesicles
Spleen
Testes
Thymus
Thyroid with parathyroids
Uterus

HISTOPATHOLOGY: Yes
The following tissues and organs were collected and placed in 10% neutral buffered formalin (except as noted):
Adrenals (2)
Aorta
Bone with marrow
Femur
Sternum
Bone marrow smear (from femur)
Brain
Cervix
Epididymides (2)
Eyes with optic nerves (2)
Gastrointestinal tract: Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum
Heart
Kidneys (2)
Larynx
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by inflation with fixative)
Lymph nodes: Axillary (2), Mandibular (2), Mesenteric
Ovaries (2) with oviducts
Pancreas
Peripheral nerve (sciatic)
Peyer’s patches
Pharynx
Pituitary
Prostate
Salivary glands (mandibular [2])
Seminal vesicles (2)
Skeletal muscle (rectus femoris)
Skin with mammary gland
Spinal cord (cervical, thoracic, lumbar)
Spleen
Testes (2)
Thymus
Thyroid (with parathyroids, [2])
Tongue
Trachea
Uterus
Urinary bladder
Vagina
Gross lesions

Microscopic examination was performed on all tissues listed above from all animals in the control and 1000 mg/kg bw/day groups at the scheduled necropsy. In addition, gross lesions were examined microscopically from all animals in the 100 and 300 mg/kg bw/day groups.
Other examinations:
None.
Statistics:
Each mean was presented with the standard deviation, standard error, and/or the number of animals used to calculate the mean.
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Body weight, body weight change, food consumption, continuous FOB, clinical pathology, and organ weight data were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance treated groups to the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test (Steel and Torrie, 1980).

All repeated measures analysis of variance statistical analyses for total and ambulatory locomotor activity counts: each analysis endpoint was analyzed, by sex and session, with a RANOVA. Factors in the model included treatment group, time interval, and the interaction of time interval and treatment group (TRT*TIME).
The monotonic dose-response relationship was evaluated using sequential linear trend tests based on ordinal spacing of dosage levels. The linear dose by time interaction (LinTrt*Time) was evaluated and, if significant at the 0.05 level, trend tests on treatment means were performed at the 0.05 level for each time interval. If the linear dose by time interaction was not significant, the trend test was conducted across the pooled time intervals for the entire session only.
Nonmonotonic dose responses were evaluated whenever no significant linear trends were detected but TRT and/or TRT*TIME interaction was significant at the 0.01 level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No mortality. Test substance-related clinical observations of clear and/or red material on various body surfaces (mouth, nose, and/or ventral neck) were noted in the 300 mg/kg/day group males and in the 1000 mg/kg/day group males and females.
Mortality:
mortality observed, treatment-related
Description (incidence):
No mortality. Test substance-related clinical observations of clear and/or red material on various body surfaces (mouth, nose, and/or ventral neck) were noted in the 300 mg/kg/day group males and in the 1000 mg/kg/day group males and females.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Potentially test substance-related lower aspartate aminotransferase (AST) and alanine aminotransferase (ALT) values were noted in the 1000 mg/kg/day group females. Changes were within the WIL Research historical control range and were not relevant.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
A difference was noted at 1000 mg/kg bw/day for the ease of removal and an overall trend towards lower totla and ambulatory activity counts in 1000 mg/kg bw/day females. Both findings were considered common findings and not treatment related.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related higher liver weights were noted in the 1000 mg/kg/day group males and females.Weight chagnes were within the WIL Research historical control range.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related hepatocyte hypertrophy was noted in the liver in the 1000 mg/kg/day group males and females
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
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:
clinical signs
mortality
dermal irritation
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
immunology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, oral administration of the test substance to Crl:CD(SD) rats for 90 or 91 consecutive days at up to 1000 mg/kg bw/day was well tolerated.
Test substance-related effects were limited to clear and/or red material on various body surfaces (mouth, nose, and/or ventral neck) in the 300 mg/kg bw/day group males and in the 1000 mg/kg bw/day group males and females, and higher liver weights and hepatocyte hypertrophy in the 1000 mg/kg bw/day group males and females. However, none of these effects were considered to be adverse. Therefore, the NOAEL (no-observed-adverse-effect level) was considered to be 1000 mg/kg bw/day, the highest dosage level administered.
Executive summary:

The test substance was administered by daily oral gavage during 90 -day to Crl:CD (SD) rats (10/sex) at dose levels of 0, 100, 300 or 1000 mg/kg bw/day.

There were no test substance-related deaths. Body weight (gain), food consumption, FOB, opthalmoscopy and clinical biochemistry were unaffected by test substance administration.

Test substance-related clinical observations of clear and/or red material on various body surfaces (mouth, nose, and/or ventral neck) were noted in the 300 mg/kg/day group males and in the 1000 mg/kg/day group males and females.

Females at 1000 mg/kg bw/day showed statistically significantly lower mean AST values (68% of control value). The mean ALT value at 1000 mg/kg bw/day was also lower than the control group (56% of control), but this difference was not statistically significant. Although the lower AST and ALT values in the 1000 mg/kg/day group females were potentially test substance-related, the mean values for these AST and ALT differences in the test substance-treated groups were all within the WIL Research historical control range and are considered not toxicologically relevant.

Test substance-related higher mean absolute and relative (to final body weight and brain weight) liver weights were noted in the 1000 mg/kg/day group males and females. However, the only statistically significant difference was mean liver weight relative to final body weight in the 1000 mg/kg/day group males (110% when compared to controls).  This significantly higher mean relative liver weight was within the WIL Research historical control range. All other increases in absolute or relative liver weight remained below 110% of controls. These weight changes were considered borderline effects, as liver hyperthrophy was seen only (inherent to increased liver weight) and the changes were not accompanied by adverse histopathological changes.

Based on the results of this study, the NOAEL was considered to be 1000 mg/kg bw/day, the highest dose level administered.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
There were three (3) well conducted studies which did not find any adverse effects up to the maximum dose of 1000 mg/kg/day.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The studies provide sufficient information of lack of hazard at maximum experimental levels (1000 mg/kg/day). Since the registration substance is meant to be used as a lubricant additive with no other possible use, the information is sufficiently protective for humans.

Additional information

Justification for classification or non-classification

Not classified in accordance to Regulation (EC) No 1272/2008 since no adverse effects were noted up to a limit dose of 1000 mg/kg/day in all three studies, including a sub-chronic 90 day repeated dose toxicity test..