Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

Endpoint summary

Description of key information

The no-observed-adverse-effect level was 1000 mg/kg when Trixene AS was administered for 14 days via oral gavage to male and female Sprague Dawley rats.

Trixene AS induced repeat dose toxicity in male and female rats at >300 mg/kg/day. The repeat dose no-observed-adverse-effect level (NOAEL) for male rats was determined to be 300 mg/kg/day due to microscopic findings (spermatid retention). The repeat dose NOAEL for female rats was considered to be 1000 mg/kg/day, the highest dose level tested.

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April 25, 2018 to July 11, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

no guideline required

Principles of method if other than guideline:

This study is a dose range finding study to support the full sub-acute repeated dose study. The Institutional Animal Care and Use Committee (IACUC) of Smithers Avanza approved the study protocol and found it to be in accordance with provisions of the USDA Animal Welfare Act, the PHS Policy on Humane Care and Use of Laboratory Animals, and the US Interagency Research Animal Committee Principles for the Utilization and Care of Research Animals.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was selected because it is a standard species for use in toxicological studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and Strain: Sprague Dawley Rat
Supplier: Charles River Breeding Labs,; Raleigh, NC
Method of Identification: Ear tag and cage card
Number of Animals Received: Male: 22, Female: 22
Number Used on Study: Male: 20, Female: 20
Age at First Dose: Male: 9-10 weeks, Female: 9-10 weeks
Weight Range at First Dose: Male: 288.1 – 353.6 grams, Female: 221.3 – 245.5 grams
Disposition of Extra Animals: Euthanized
Animals were acclimated to laboratory conditions for at least five days prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number that was recorded on each cage label.

Husbandry Information
Feed: Certified Global Teklad Laboratory Diet 2018 (pellets) was provided ad libitum, unless otherwise noted.
Water: Filtered water was provided ad libitum via an automatic watering system supplemented with water bottles/bowls as needed.
Bedding: Certified Sani Chips® hardwood bedding
Housing: Animals were housed in one room in polycarbonate cages suspended on stainless steel racks. Each cage was affixed with a cage card containing pertinent animal and study information. Animals were partnered within the same sex and group with 2-3 animals housed together.
Temperature Range: 20 to 26 °C
Humidity Range: 30 to 70%
Light Cycle: 12-hour light/12-hour dark
Air Changes: Minimum of 10 air changes per hour

The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The water is routinely analyzed for contaminants and specific microbes. The bedding was analyzed by the manufacturer for acceptable levels of heavy metals, aflatoxins, bacteria, yeasts, molds, and organophosphates prior to certification. No contaminants were known to be present in the feed, water, or bedding at levels that might have interfered with achieving the objectives of the study.

Route of administration:

oral: gavage

Details on route of administration:

The animals were dosed via oral gavage at a volume of 2 mL/kg for 14 days. Dosing volumes were based on the animals’ most recent body weights. The first day of dosing was designated as SD 1 for each animal.

Vehicle:

peanut oil

Details on oral exposure:

Vehicle/Control Substance
Name: Peanut oil
Lot/Batch No.: 0280-0588
Supplier: Welsh, Holme & Clark Co., Inc., Newark, NJ
Purity: Assumed 100%

The neat test and vehicle/control substance were considered 100% pure for formulation purposes.
The vehicle/control substance, peanut oil, was used as received; no formulations were necessary.
For the test substance, Trixene AS, formulations were prepared daily as described in the following paragraph. All prepared formulations were maintained at room temperature until used for further formulating or for dosing.
Group 4 (500 mg/mL) formulations were prepared by adding the appropriate amount of test substance to the required amount of the vehicle/control substance until the required final volume was reached and the formulations were inverted approximately 30 times. Group 3 formulations (150 mg/mL) were prepared by mixing together the required amount of Group 4 formulations with the required amount of vehicle/control substance until the required final volume was reached and the formulations were inverted approximately 30 times. Group 2 formulations (50 mg/mL) were prepared by mixing together the required amount of Group 3 formulations with the required amount of vehicle/control substance until the required final volume was reached and the formulations were inverted approximately 30 times.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

The animals were dosed via oral gavage at a volume of 2 mL/kg for 14 days.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per dose group (5 male/5 female)

Control animals:

yes, concurrent vehicle

Details on study design:

This study was designed to use the fewest number of animals possible, consistent with the objective of the study, the scientific needs of the Sponsor, and contemporary scientific standards.
The oral route was selected because it is the most appropriate route to determine the toxicological effects of the test substance.
The dose levels were selected based on the results from previous toxicology studies performed by the Sponsor or Sponsor’s designee for a similar multi-constituent substance. A single dose acute oral gavage study was performed with undiluted substance at up to 5 mL/kg. Yellow mucoid diarrhea was seen in all the male animals and two females 3 hours after dosing. No other clinical signs were observed and no mortality occurred.

Positive control:

Not required

Observations and examinations performed and frequency:

Animal Observations/Measurements (Procedure: Frequency of Testing)
Physical Examinations: Study Day 1 (prior to irritation of dosing); Daily thereafter; Prior to necropsy
Cageside Observations: ≥ 2 times daily
Body Weights: Study Day 1 (prior to initiation of dosing); Study Day 4 and 8; At the time of unscheduled termination; Prior to necropsy
Food Consumption: Study Day 1-4, 4-8 and 8-15
Cageside observations included observation for mortality, moribundity, general health, and signs of toxicity. Physical examinations included evaluation of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, and somatomotor and behavior patterns.

Sacrifice and pathology:

Termination
Moribund animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.
On SD 15, all surviving animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.

Necropsy
Animals were necropsied as soon as possible after the time of death. Animals were necropsied, bone marrow smears were prepared, required organs were weighed, and protocol-specified tissues were collected and preserved. No organ weights were collected from animals euthanized prior to scheduled termination.
Gross necropsy included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. Organ weights were collected as soon as possible after dissection and paired organs were weighed together. Tissues were preserved in 10% neutral buffered formalin (NBF) with the exception of the eyes (and associated ocular tissue) and testes (with epididymides), which were preserved in modified Davidson’s fixative and subsequently transferred to 10% NBF. Two bone marrow smears were prepared from the left femur and the slides were air-dried, fixed in methanol, and stored at room temperature for possible future evaluation. No analysis of the bone marrow smears was deemed necessary; therefore, the unstained slides were discarded prior to report finalization.

Statistics:

Electronic data collection, including randomization, dose formulations and dispensing, dosing, animal husbandry, environmental enrichment, physical examinations, cageside observations, body weights, body weight changes, food consumption, organ weights, and gross pathology was performed using Provantis™ Version 8 (Instem LSS, Limited; Stone, UK). Environmental monitoring was performed using Rees Environmental Monitoring System (Rees Scientific Corporation; Trenton, NJ
Descriptive statistics (mean, standard deviation, and N) are presented in the data appendices for all quantitative measurement data.

Clinical signs:

no effects observed

Description (incidence and severity):

Treatment with Trixene AS had no effect on mortality. On SD 12 Group 4 female 25991 (1000 mg/kg/day) was observed with red discharge from oral cavity prior to dosing. The source of the hemorrhage could not be identified and was suspected to be the result of a rupture or tear in a sublingual vessel, possibly due to a lower incisor puncturing the tongue. This animal was euthanized as moribund due to humane concerns. This was not test substance-related because it it was noted in a single animal without evidence of a dose-response.
Treatment with Trixene AS had no effect on physical examinations or cageside observations. With the exception of Group 4 female 2599, no other abnormalities were observed in any of the animals throughout the study.
Group 4 female 25991 (1000 mg/kg/day) had frank, active hemorrhage from oral cavity. The veterinarian suspected a rupture or tear in the sublingual vessel potentially due to a lower incisor puncturing the tongue. Hemorrhage was limited to oral cavity. Nasal epistaxis was not present. The animal was not dosed and was euthanized as moribund on SD 12 due to humane concerns.

Mortality:

no mortality observed

Description (incidence):

Treatment with Trixene AS had no effect on mortality.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment with Trixene AS had no effect on body weights or body weight changes. No significant differences were observed in mean body weights or total mean body weight changes (SD 1 to 15). From SD 1 to 4, the mean body weight changes for Group 3 (300 mg/kg/day) males and Group 4 (1000 mg/kg/day) males and females were significantly lower when compared to the control. The significant difference during this interval appeared to be related to the initiation of dosing and palatability of the test substance and was not adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Treatment with Trixene AS had no effect on food consumption. Animals were group housed (2 to 3 per cage) and therefore there were only two cages per group that could be assessed. Thus, although the mean total food consumption (SD 1-15) was lower in treated groups compared to the control, and was significantly lower in Group 2 (100 mg/kg/day) females and Group 3 (300 mg/kg/day) males this was variable between cages and appeared to be inconsistent in response and therefore unrelated to dose level. The mean food consumption was significantly lower in Group 2 males (SD 1-4) and females (SD 4-8, 8-15), Group 3 males (SD 1-4, 8 -15) and females (SD 1-4, 4-8) and Group 4 males (SD 1-4) and females (SD 1-4, 4-8). Additionally, no significant correlations in body weight changes were observed. Therefore the effects on food consumption were not considered adverse.

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treatment with Trixene AS had an effect on organ weights. The absolute and relative spleen weights of Group 4 (1000 mg/kg/day) males was significantly increased when compared to control. A trend toward higher absolute and relative spleen weight was noted in Group 2 and 2 males but the increase was not statistically significant. Although the effect was dose-related it was not considered adverse in the absence of any other observations and only occurring in one sex.
All other significant differences in organ weights were considered incidental and not related to the test substance, Trixene AS because they were isolated, occurred in a single sex, and were not dose-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Treatment with Trixene AS had no effect on gross pathology findings. No observations were noted in all animals surviving to scheduled necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology findings, and absolute and relative organ weights.
Treatment with Trixene AS at doses up to 1000 mg/kg had no adverse effect on the parameters evaluated.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect on the parameters evaluated

Critical effects observed:

no

Animal Disposition and Physical Examinations

Day Numbers Relative to Start Date

Group

Sex

Animal

Clinical Sign

Site

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

1

M

25956

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25957

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25958

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25959

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25960

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

2

M

25966

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25967

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25968

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25969

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25970

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

3

M

25976

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25977

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25978

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25979

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25980

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

4

M

25896

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25987

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25988

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25989

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25990

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

1

F

25961

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25962

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25963

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25964

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25965

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

2

F

25971

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25972

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25973

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25974

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25975

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

3

F

25981

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25982

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25983

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25984

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25985

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

4

F

25991

No Abnormalities Detected Discharge Moribund Kill

Mouth

X - -

X - -

X - -

X - -

X - -

X - -

X - -

X - -

X - -

X - -

X - -

X D X

- - -

- - -

- - -

25992

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25993

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25994

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

25995

No Abnormalities Detected Terminal Kill

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X -

X X

Nominal Dose: Group 1 – 0 mg/kg/day         Group 2 – 100 mg/kg/day Group 3 – 300 mg/kg/day Group 4 – 1000 mg/kg/day

 

Cageside Observations

No abnormalities were observed in any of the animals from SD 1 through the end of the study.

 

Veterinarian Examinations/Treatments

Day Numbers Relative to Start Date
Group	Sex	Animal	Veterinarian Examination Summary	Treatments	Study Days
4	F	25991	Animal had frank, active hemorrhage from oral cavity. Suspected rupture or tear in a sublingual vessel if a lower incisor punctured the tongue. Hemorrhage was limited to oral cavity. Nasal epistaxis was not present. The animal was not dosed.	Euthanized due to humane concerns	SD 12

Veterinarian exam day = first treatment day unless otherwise noted.

Note: All conditions/observations were resolved or resolving by the end of the specified treatment period unless noted otherwise.

 

Body Weights (g)

Day Numbers Relative to Start Date
Group	Sex	Animal	1	4	8	15	Group	Sex	Animal	1	4	8	12	15
1	M	25956 25957 25958 25959 25960	331.3 323.7 336.8 343.1 288.1	344.3 340.0 361.6 372.1 308.8	396.5 364.8 361.6 410.6 331.1	398.1 387.4 440.6 461.2 360.4	3	M	25976 25977 25978 25979 25980	322.8 316.1 313.6 336.8 325.7	332.7 324.4 319.2 354.7 341.6	350.2 342.9 347.3 379.4 369.7	- - - - -	378.2 360.4 375.3 408.1 401.5
		Mean S.D. N	324.60 21.62 5	345.36 24.21 5	372.92 31.30 5	409.54 40.83 5			Mean S.D. N	323.00 9.13 5	334.52 14.12 5	357.90 15.80 5	- - 0	384.70 19.69 5
2	M	25966 25967 25968 25969 25970	325.4 323.5 329.3 323.1 326.0	338.0 343.9 338.9 335.5 341.2	350.1 366.3 363.8 366.7 377.2	363.5 389.2 396.2 415.8 417.1	4	M	25986 25987 25988 25989 25990	353.6 328.2 320.6 336.6 322.3	369.9 335.5 323.5 343.3 331.9	399.2 358.8 349.1 378.9 358.8	- - - - -	452.2 402.2 378.8 421.1 395.6
		Mean S.D. N	325.46 2.47 5	339.50 3.20 5	364.82 9.70 5	396.36 22.02 5			Mean S.D. N	332.26 13.47 5	340.82 17.75 5	368.96 20.09 5	- - 0	409.98 28.04 5
1	F	25961 25962 25963 25964 25965	234.2 229.3 245.5 221.3 241.1	249.0 233.7 266.6 217.4 247.3	263.1 247.8 282.7 222.5 263.4	272.3 248.3 311.4 238.6 278.4	3	F	25981 25982 25983 25984 25985	229.5 241.6 239.8 238.3 234.4	235.6 246.3 240.0 245.0 231.9	242.5 251.1 241.2 260.0 247.6	- - - - -	257.8 267.2 263.4 276.9 251.2
		Mean S.D. N	234.28 9.56 5	242.80 18.39 5	255.90 22.40 5	269.80 28.49 5			Mean S.D. N	236.72 4.83 5	239.76 6.11 5	248.48 7.57 5	- - 0	263.30 9.71 5
2	F	25971 25972 25973 25974 25975	230.9 244.8 232.4 234.2 229.8	232.6 242.4 232.6 236.6 235.0	244.4 243.3 241.2 245.4 239.7	251.8 271.2 262.7 248.4 265.6	4	F	25991 25992 25993 25994 25995	232.3 240.1 222.2 222.6 231.5	222.5 235.8 226.2 218.7 233.4	241.3 249.4 235.2 227.4 245.8	242.2 - - - -	- 280.9 251.7 251.7 256.7
		Mean S.D. N	234.42 6.03 5	235.84 4.04 5	242.80 2.33 5	259.94 9.56 5			Mean S.D. N	229.74 7.50 5	227.32 7.21 5	239.82 8.74 5	242.20 - 1	260.25 13.97 4

Nominal Dose: Group 1 – 0 mg/kg/day         Group 2 – 100 mg/kg/day Group 3 – 300 mg/kg/day Group 4 – 1000 mg/kg/day

 

Body Weight Changes (g)

Day Numbers Relative to Start Dates
Group	Sex	Animal	From: To:	1 4	4 8	8 12	8 15	Absolute Change	Percent Change
								1 15	1 15
1	M	25956 25957 25958 25959 25960		13.0 16.3 24.8 29.0 20.7	52.2 24.8 0.0 38.5 22.3	- - - - -	1.6 22.6 79.0 50.6 29.3	66.8 63.7 103.8 118.1 72.3	20.16 19.68 30.82 34.42 25.10
		Mean S.D. N		20.76 6.41 5	27.56 19.50 5	- - -	36.62 29.45 5	84.94 24.47 5	26.04 6.50 5
2	M	25966 25967 25968 25969 25970		12.6 20.4 9.6 12.4 15.2	12.1 22.4 24.9 31.2 36.0	- - - - -	13.4 22.9 32.4 49.1 39.9	38.1 65.7 66.9 92.7 91.1	11.71 20.31 20.31 28.69 27.94
		Mean S.D. N		14.04 4.07 5	25.32 9.11 5	- - -	31.54 13.99 5	70.90 22.37 5	21.79 6.92 5
3	M	25976 25977 25978 25979 25980		9.9 8.3 5.6 17.9 15.9	17.5 18.5 28.1 24.7 28.1	- - - - -	28.0 17.5 28.0 28.7 31.8	55.4 44.3 61.7 71.3 75.8	17.16 14.01 19.67 21.17 23.27
		Mean S.D. N		11.52* 5.19 5	23.38 5.12 5	- - -	26.80 5.43 5	61.70 12.58 5	19.06 3.59 5
4	M	25986 25987 25988 25989 25990		16.3 7.3 2.9 6.7 9.6	29.3 23.3 25.6 35.6 26.9	- - - - -	53.0 43.4 29.7 42.2 36.8	98.6 74.0 58.2 84.5 73.3	27.88 22.55 18.158 25.10 22.74
		Mean S.D. N		8.56* 4.95 5	28.14 4.70 5	- - -	41.02 8.61 5	77.72 14.97 5	23.29 3.59 5
1	F	25961 25962 25963 25964 25965		14.8 4.4 21.1 -3.9 6.2	14.1 14.1 16.1 5.1 16.1	- - - - -	9.2 0.5 28.7 16.1 15.0	38.1 19.0 65.9 17.3 37.3	16.27 8.29 26.84 7.82 15.47
		Mean S.D. N		8.52 9.67 5	13.10 4.58 5	- - -	13.90 10.33 5	35.52 19.61 5	14.94 7.73 5
2	F	25971 25972 25973 25974 25975		1.7 -2.4 0.2 2.4 5.2	11.8 0.9 8.6 8.8 4.7	- - - - -	7.4 27.9 21.5 3.0 25.9	20.9 26.4 30.3 14.2 35.8	9.05 10.78 13.04 6.06 15.58
		Mean S.D. N		1.42 2.80 5	6.96 4.22 5	- - -	17.14 11.25 5	25.52 8.35 5	10.90 3.65 5
3	F	25981 25982 25983 25984 25985		6.1 4.7 0.2 6.7 -2.5	6.9 4.8 1.2 15.0 15.7	- - - - -	15.3 16.1 22.2 16.9 3.6	28.3 25.6 23.6 38.6 16.8	12.33 10.60 9.84 16.20 7.17
		Mean S.D. N		3.04 4.01 5	8.72 6.39 5	- - -	14.82 6.83 5	26.58 7.95 5	11.23 3.34 5
4	F	25991 25992 25993 25994 25995		-9.8 -4.3 4.0 -3.9 1.9	18.8 13.6 9.0 8.7 12.4	0.9 - - - -	- 31.5 16.5 24.3 10.9	- 40.9 29.5 29.1 25.2	- 16.99 13.28 13.07 10.89
		Mean S.D. N		-2.42* 5.48 5	12.50 4.11 5	0.90 - 1	20.80 9.00 4	31.15 6.72 4	13.56 2.53 4

* - Significantly different from the control value, p ≤ 0.05

Nominal Dose: Group 1 – 0 mg/kg/day         Group 2 – 100 mg/kg/day Group 3 - 300 mg/kg day  Group 4 – 1000 mg/kg/day

 

Gross Pathology Observations

Animal Ref.	Mode of Death	Death		Observation (s)
		Day	(Week)
Group 1: Male
25956 25957 25958 25959 25960	TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL	15 15 15 15 15	(3) (3) (3) (3) (3)	No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions
Group 2: Male
25966 25967 25968 25969 25970	TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL	15 15 15 15 15	(3) (3) (3) (3) (3)	No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions
Group 3: Male
25976 25977 25978 25979 25980	TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL	15 15 15 15 15	(3) (3) (3) (3) (3)	No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions
Group 4: Male
25986 25987 25988 25989 25990	TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL	15 15 15 15 15	(3) (3) (3) (3) (3)	No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions
Group 1: Female
25961 25962 25963 25964 25965	TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL	15 15 15 15 15	(3) (3) (3) (3) (3)	No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions
Group 2: Female
25971 29572 25973 25974 25975	TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL	15 15 15 15 15	(3) (3) (3) (3) (3)	No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions
Group 3: Female
25981 25982 25983 25984 25985	TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL	15 15 15 15 15	(3) (3) (3) (3) (3)	No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions
Group 4: Female
25991       25992 25993 25994 29995	MORIBUND KILL       TERMINAL KILL TERMINAL KILL TERMINAL KILL TERMINAL KILL	12       15 15 15 15	(2)       (3) (3) (3) (3)	Esophagus: NO PERFORATION DETECTED Trachea: NO PERFORATION DETECTED Mouth; no correlate with clinical observations Any remaining protocol required tissues, which have been examined, have no visible lesions No Visible Lesions No Visible Lesions No Visible Lesions No Visible Lesions

Nominal Dose:    Group 1 – 0 mg/kg/day                      Group 2 – 100 mg/kg/day Group 3 – 300 mg/kg/day Group 4 – 1000 mg/kg/day

 

Organ Weight Data

Day 15 Relative to Start Date
Group	Sex	Animal	TBW g	--- Brain ---		---- Adrenal Gland ----			---- Heart ----
				Abs	/BW	Abs	/BW	/BR	Abs	/BW	/BR
1	M	25956 25957 25958 25959 25960	398.1 387.4 440.6 461.2 360.4	2.104 2.092 2.041 2.099 2.139	0.5285 0.5400 0.4632 0.4551 0.5935	0.0507 0.0590 0.0552 0.0359 0.0529	0.0127 0.0152 0.0125 0.0078 0.0147	0.0241 0.0282 0.0270 0.0171 0.0247	1.435 1.390 1.459 1.498 1.243	0.3605 0.3588 0.3311 0.3248 0.3449	0.6820 0.6644 0.7148 0.7137 0.5811
		Mean S.D. N	409.54 40.83 5	2.0950 0.0352 5	0.51606 0.05752 5	0.05074 0.00885 5	0.02158 0.00293 5	0.02422 0.00432 5	1.4050 0.0987 5	0.34402 0.01602 5	0.67120 0.05475 5
2	M	25966 25967 25968 25969 25970	363.5 389.2 396.2 415.8 417.1	1.877 1.988 1.982 2.061 2.241	0.5164 0.5108 0.5003 0.4957 0.5373	0.0322 0.0514 0.0605 0.0359 0.0594	0.0089 0.0132 0.0153 0.0086 0.0142	0.0172 0.0259 0.0305 0.0174 0.0265	1.272 1.277 1.607 1.588 1.577	0.3499 0.3281 0.4056 0.3747 0.3781	0.6777 0.6424 0.8108 0.7559 0.7037
		Mean S.D. N	396.36 22.02 5	2.0298 0.1351 5	0.51210 0.01631 5	0.04788 0.01317 5	0.01204 0.00310 5	0.02350 0.00593 5	1.4582 0.1686 5	0.36728 0.02949 5	0.71810 0.06632 5
3	M	25976 25977 25978 25979 25980	378.2 360.4 375.3 408.1 401.5	1.917 2.145 2.084 2.153 2.157	0.5069 0.5952 0.5553 0.5276 0.5372	0.0556 0.0552 0.0432 0.0518 0.0552	0.0147 0.0153 0.0115 0.0127 0.0137	0.0290 0.0257 0.0207 0.0241 0.0256	1.257 1.294 1.357 1.302 1.301	0.3224 0.3590 0.3616 0.3190 0.3240	0.6557 0.6033 0.6512 0.6047 0.6032
		Mean S.D. N	384.70 19.69 5	2.0912 0.1018 5	0.54444 0.03332 5	0.05220 0.00526 5	0.01358 0.00153 5	0.02502 0.00301 5	1.3022 0.0358 5	0.33920 0.01987 5	0.62362 0.02728 5
4	M	25986 25987 25988 25989 25990	452.2 402.2 378.8 421.1 395.6	2.189 2.163 2.085 2.076 2.099	0.4841 0.5378 0.5504 0.4930 0.5306	0.0675 0.0637 0.0524 0.0603 0.0518	0.0149 0.0158 0.0138 0.0143 0.0131	0.0308 0.0294 0.0251 0.0290 0.0247	1.768 1.374 1.361 1.602 1.211	0.3910 0.3416 0.3593 0.3804 0.3061	0.8077 0.6352 0.6528 0.7717 0.5769
		Mean S.D. N	409.98 28.04 5	2.1224 0.0505 5	0.51918 0.02902 5	0.05914 0.00692 5	0.01438 0.00103 5	0.02780 0.00273 5	1.4632 0.2203 5	0.35568 0.03365 5	0.68876 0.09708 5

 

Day 15 Relative to Start Date
Group	Sex	Animal	---- Kidneys ----			---- Liver ----			---- Pituitary ----
			Abs	/BW	/BR	Abs	/BW	/BR	Abs	/BW	/BR
1	M	25956 25957 25958 25959 25960	2.705 2.746 3.135 3.316 2.950	0.6795 0.7088 0.7115 0.7190 0.8185	1.2856 1.3126 1.5360 1.5798 1.3791	16.590 14.621 17.247 18.853 13.314	4.1673 3.7741 3.9144 4.0878 3.6942	7.8850 6.9890 8.4503 8.9819 6.2244	0.0087 0.0087 0.0096 0.0122 0.0100	0.0022 0.0022 0.0022 0.0026 0.0028	0.0041 0.0042 0.0047 0.0058 0.0047
		Mean S.D. N	2.9704 0.2587 5	0.72746 0.05306 5	1.41862 0.13253 5	16.1250 2.1841 5	3.92756 0.20083 5	7.70612 1.10941 5	0.00984 0.00144 5	0.00240 0.00028 5	0.00470 0.00067 5
2	M	25966 25967 25968 25969 25970	2.740 2.834 2.860 3.077 2.534	0.7538 0.7282 0.7219 0.7400 0.6075	1.4598 1.4256 1.4430 1.4930 1.1307	14.122 14.950 14.901 18.672 15.092	3.8850 3.8412 3.7610 4.4906 3.6183	7.5237 7.5201 7.5182 9.0597 6.7345	0.0085 0.0101 0.0101 0.0099 0.0102	0.0023 0.0026 0.0025 0.0024 0.0024	0.0045 0.0051 0.0051 0.0048 0.0046
		Mean S.D. N	2.8090 0.1971 5	0.71028 0.05873 5	1.39042 0.14730 5	15.5474 1.7872 5	3.91922 0.33516 5	7.67124 0.84755 5	0.00976 0.00071 5	0.00244 0.00011 5	0.00482 0.00028 5
3	M	25976 25977 25978 25979 25980	3.059 2.239 2.564 2.657 2.818	0.8088 0.6213 0.6832 0.6511 0.7019	1.5957 1.0438 1.2303 1.2341 1.3064	13.970 12.568 13.662 14.252 15.879	3.6938 3.4872 3.6403 3.4923 3.9549	7.2874 5.8592 6.5557 6.6196 7.3616	0.0071 0.0073 0.0098 0.0105 0.0095	0.0019 0.0020 0.0026 0.0026 0.0024	0.0037 0.0034 0.0047 0.0049 0.0044
		Mean S.D. N	2.6674 0.3043 5	0.69326 0.07156 5	1.28206 0.02244 5	14.0662 1.1977 5	3.65370 0.19123 5	6.73670 0.61446 5	0.00884 0.00154 5	0.00230 0.00033 5	0.00422 0.00065 5
4	M	25986 25987 25988 25989 25990	3.117 2.703 2.919 2.803 2.748	0.6893 0.6721 0.7706 0.6656 0.9646	1.4239 1.2497 1.4000 1.3502 1.3092	19.537 15.372 15.263 17.446 15.815	4.3204 3.8820 4.0293 4.1430 3.9977	8.9251 7.1068 8.3204 8.4037 7.5345	0.0123 0.0038 0.0073 0.0122 0.0106	0.0027 0.0009 0.0019 0.0029 0.0027	0.0056 0.0018 0.0035 0.0059 0.0051
		Mean S.D. N	2.8580 0.1658 5	0.69844 0.04206 5	1.34660 0.07005 5	16.6866 1.8177 5	4.06248 0.18448 5	7.85810 0.77370 5	0.00924 0.00365 5	0.00222 0.00083 5	0.00438 0.00171 5

 

Day 15 Relative to Start Date
Group	Sex	Animal	---- Spleen ----			---- Thymus ----			---- Thyroid ----
			Abs	/BW	/BR	Abs	/BW	/BR	Abs	/BW	/BR
1	M	25956 25957 25958 25959 25960	0.514 0.931 0.705 0.973 0.733	0.1291 0.2403 0.1600 0.2110 0.2034	0.2443 0.4450 0.3554 0.4636 0.3427	0.431 0.493 0.556 0.702 0.418	0.1083 0.1273 0.1262 0.1522 0.1160	0.2048 0.2357 0.2724 0.3344 0.1954	0.0214 0.0176 0.0325 0.0247 0.0196	0.0054 0.0045 0.0074 0.0054 0.0054	0.0102 0.0084 0.0159 0.0118 0.0092
		Mean S.D. N	0.7712 0.1859 5	0.18876 0.04402 5	0.36820 0.08877 5	0.5200 0.1156 5	0.12600 0.01659 5	0.24854 0.05666 5	0.02316 0.00584 5	0.00562 0.00107 5	0.01110 0.00297 5
2	M	25966 25967 25968 25969 25970	0.574 0.874 0.961 1.067 1.027	0.1579 0.2246 0.2426 0.2566 0.2462	0.3058 0.4396 0.4849 0.5177 0.4583	0.436 0.447 0.599 0.664 0.564	0.1199 0.1149 0.1512 0.1597 0.1352	0.2323 0.2248 0.3022 0.3222 0.2517	0.0250 0.0233 0.0228 0.0207 0.0221	0.0069 0.0060 0.0058 0.0050 0.0053	0.0133 0.0117 0.0115 0.0100 0.0099
		Mean S.D. N	0.9006 0.1967 5	0.22558 0.03956 5	0.44126 0.08123 5	0.5420 0.0986 5	0.13618 0.01935 5	0.26664 0.04331 5	0.02278 0.00158 5	0.00580 0.00073 5	0.01128 0.00140 5
3	M	25976 25977 25978 25979 25980	0.868 1.029 0.852 1.116 0.855	0.2295 0.2855 0.2270 0.2735 0.2130	0.4528 0.4797 0.4088 0.5183 0.3964	0.606 0.449 0.554 0.563 0.522	0.1602 0.1246 0.1476 0.1380 0.1300	0.3161 0.2093 0.2658 0.2615 0.2420	0.0227 0.0204 0.0236 0.0208 0.0159	0.0060 0.0057 0.0063 0.0051 0.0040	0.0118 0.0095 0.0113 0.0097 0.0074
		Mean S.D. N	0.9440 0.1214 5	0.24570 0.03177 5	0.45120 0.05029 5	0.5388 0.0585 5	0.14008 0.01420 5	0.25894 0.03896 5	0.02068 0.00298 5	0.00542 0.00091 5	0.00994 0.00173 5
4	M	25986 25987 25988 25989 25990	1.201 0.995 0.934 1.224 1.163	0.2656 0.2474 0.2466 0.2907 0.2940	0.5487 0.4600 0.4480 0.5896 0.5541	0.654 0.616 0.411 0.596 0.510	0.1446 0.1532 0.1085 0.1415 0.1289	0.2988 0.2848 0.1971 0.2871 0.2430	0.0224 0.0232 0.0225 0.0214 0.0251	0.0050 0.0058 0.0059 0.0051 0.0063	0.0102 0.0107 0.0108 0.0103 0.0120
		Mean S.D. N	1.1034* 0.1305 5	0.26886* 0.02278 5	0.52008* 0.06248 5	0.5574 0.0974 5	0.13534 0.01735 5	0.26216 0.04206 5	0.02292 0.00138 5	0.00562 0.00055 5	0.01080 0.00072 5

 

Day 15 Relative to Start Date
Group	Sex	Animal	---- Prostate ----			---- Seminal Vesicles ----			---- Testes ----
			Abs	/BW	/BR	Abs	/BW	/BR	Abs	/BW	/BR
1	M	25956 25957 25958 25959 25960	0.572 0.691 0.888 1.044 1.020	0.1437 0.1784 0.2015 0.2264 0.2830	0.2719 0.3303 0.4351 0.4971 0.4769	1.330 1.238 1.334 0.801 1.136	0.3341 0.3196 0.3028 0.1737 0.3152	0.6321 0.5918 0.6536 0.3816 0.5311	4.484 4.735 4.427 5.343 4.451	1.1264 1.2223 1.0048 1.1585 1.2350	2.1312 2.2634 2.1690 2.5455 2.0809
		Mean S.D. N	0.8430 0.2063 5	0.20660 0.05246 5	0.40232 0.09731 5	1.1678 0.2205 5	0.28908 0.06546 5	0.55804 0.10910 5	4.6880 0.3864 5	1.14940 0.09242 5	2.23800 0.18442 5
2	M	25966 25967 25968 25969 25970	0.931 0.886 0.874 1.234 1.071	0.2561 0.2276 0.2206 0.2968 0.2568	0.4960 0.4457 0.4410 0.5987 0.4779	1.165 1.173 1.201 1.192 1.000	0.3205 0.3014 0.3031 0.2867 0.2398	0.6207 0.5900 0.6060 0.5784 0.4462	3.964 4.005 3.613 4.839 2.942	1.0905 1.0290 0.9119 1.1638 0.7053	2.1119 2.0146 1.8229 2.3479 1.312
		Mean S.D. N	0.9992 0.1528 5	0.25158 0.03011 5	0.49186 0.06392 5	1.1462 0.0830 5	0.29030 0.03067 5	0.56826 0.07009 5	3.8726* 0.6880 5	0.98010 0.17923 5	1.92202 0.38954 5
3	M	25976 25977 25978 25979 25980	0.620 1.064 1.021 0.949 0.773	0.1639 0.2952 0.2720 0.2325 0.1925	0.3234 0.4960 0.4899 0.4408 0.3584	0.944 1.045 1.440 0.896 1.285	0.2496 0.2900 0.3837 0.2196 0.3200	0.4924 0.4872 0.6910 0.4162 0.5957	5.028 4.331 4.172 4.049 3.808	1.3295 1.2017 1.1124 0.9922 0.9484	2.6228 2.0191 2.0034 1.8806 1.7654
		Mean S.D. N	0.8854 0.1853 5	0.23122 0.05429 8	0.42170 0.07779 5	1.1220 0.2326 5	0.29258 0.06371 5	0.53650 0.10750 5	4.2782 0.4608 5	1.11684 0.15520 5	2.05826 0.33188 5
4	M	25986 25987 25988 25989 25990	1.021 0.800 0.826 1.210 0.868	0.2258 0.1989 0.2181 0.2873 0.2194	0.4664 0.3699 0.3962 0.5829 0.4135	1.198 1.478 1.143 0.942 1.352	0.2649 0.3675 0.3017 0.2237 0.3418	0.5473 0.6833 0.5482 0.4538 0.6441	4.674 4.529 4.002 4.312 3.919	1.0336 1.1261 1.0565 1.0240 0.9906	2.1352 2.0939 1.9194 2.0771 1.8671
		Mean S.D. N	0.9450 0.1711 5	0.22990 0.03362 5	0.44578 0.08439 5	1.2226 0.2047 5	0.29992 0.05779 5	0.57534 0.09039 5	4.2872 0.3262 5	1.04616 0.05058 5	2.01854 0.11777 5

* - Significantly different from the control value, p ≤ 0.05

Nominal Dose:   Group 1 – 0 mg/kg/day                      Group 2 – 100 mg/kg/day Group 3 – 300 mg/kg/day Group 4 – 1000 mg/kg/day

 

Organ Weight Data (continued)

Day 15 Relative to Start Date
Group	Sex	Animal	TBW g	--- Brain ---		---- Adrenal Gland ----			---- Heart ----
				Abs	/BW	Abs	/BW	/BR	Abs	/BW	/BR
1	F	25961 25962 25963 25964 29565	272.3 248.3 311.4 238.6 278.4	1.947 2.105 2.000 1.898 1.962	0.7150 0.8478 0.6423 0.7955 0.7047	0.0590 0.525 0.0682 0.0760 0.0742	0.0217 0.0211 0.0219 0.0319 0.0267	0.0303 0.0249 0.0341 0.0400 0.0378	1.108 0.972 1.083 0.817 1.079	0.4069 0.3915 0.3478 0.3424 0.3876	0.5691 0.4618 0.5415 0.4305 0.5499
		Mean S.D. N	269.50 28.49 5	1.9824 0.0777 5	0.74106 0.08080 5	0.06598 0.01004 5	0.02466 0.00463 5	0.03342 0.00603 5	1.0118 0.1208 5	0.37524 0.02851 5	0.51056 0.06066 5
2	F	25971 25972 25973 25974 25975	251.8 271.2 262.7 248.4 265.6	2.007 2.051 2.082 1.997 1.972	0.7971 0.7563 0.7925 0.8039 0.7425	0.0437 0.0594 0.0601 0.0622 0.0724	0.0174 0.0219 0.0229 0.0250 0.0273	0.028 0.0290 0.0289 0.0311 0.0367	0.989 1.042 1.098 0.943 1.156	0.3928 0.3842 0.4180 0.3796 0.4352	0.4928 0.5080 0.5274 0.4722 0.5862
		Mean S.D. N	259.94 9.56 5	2.0218 0.0441 5	0.77846 0.02728 5	0.05956 0.01029 5	0.02290 0.00371 5	0.02950 0.00535 5	1.0456 0.0847 5	0.40196 0.02378 5	0.51732 0.04350 5
3	F	25981 25982 25983 29584 29585	257.8 267.2 263.4 276.9 251.2	2.090 2.005 1.953 2.034 1.973	0.8107 0.7504 0.7415 0.7346 0.7854	0.0716 0.0576 0.0503 0.0595 0.0579	0.0278 0.0216 0.0191 0.0215 0.0230	0.0343 0.0287 0.0258 0.0293 0.0293	1.003 1.161 1.002 1.087 1.070	0.3891 0.4345 0.3804 0.3926 0.4260	0.4799 0.5791 0.5131 0.5344 0.5423
		Mean S.D. N	263.30 9.71 5	2.0110 0.0539 5	0.76452 0.03238 5	0.05938 0.00770 5	0.02260 0.00323 5	0.02948 0.00306 5	1.0646 0.0662 5	0.40452 0.02409 5	0.52976 0.03666 5
4	F	25992 25993 25994 25995	280.9 251.7 251.7 256.7	1.667 2.077 2.023 2.049	0.5934 0.8252 0.8037 0.7892	0.0671 0.0681 0.0661 0.0673	0.0239 0.0271 0.0263 0.0262	0.0403 0.0328 0.0327 0.0328	1.030 0.853 1.180 1.171	0.3667 0.3389 0.4688 0.4562	0.6179 0.4107 0.5833 0.5715
		Mean S.D. N	260.25 13.97 4	1.9540 0.1926 4	0.75513 0.10844 4	0.06715 0.00082 4	0.02588 0.00138 4	0.03465 0.00377 4	1.0585 0.1533 4	0.40765 0.06455 4	0.54585 0.09223 4

 

Day 15 Relative to Start Date
Group	Sex	Animal	---- Kidneys ----			---- Liver ----			---- Pituitary ----
			Abs	/BW	/BR	Abs	/BW	/BR	Abs	/BW	/BR
1	F	25961 25962 25963 25964 29565	1.950 1.628 2.300 1.849 2.067	0.7161 0.6557 0.7386 0.7749 0.7425	1.0015 0.7734 1.1500 0.9742 1.0535	10.199 9.232 13.659 8.394 9.558	3.7455 3.7181 4.3863 3.5180 3.4332	5.2383 4.3857 6.8295 4.4226 4.8716	0.0129 0.0169 0.0137 0.0087 0.0116	0.0047 0.0068 0.0044 0.0036 0.0042	0.0066 0.0080 0.0069 0.0046 0.0059
		Mean S.D. N	1.9588 0.2498 5	0.72556 0.04433 5	0.99052 0.13868 5	10.2084 2.0356 5	3.76022 0.37403 5	5.14954 1.00241 5	0.01276 0.00299 5	0.00474 0.00122 5	0.00640 0.00126 5
2	F	25971 25972 25973 25974 25975	1.782 1.896 2.287 1.798 2.072	0.7077 0.6991 0.8706 0.7238 0.7801	0.8879 0.9244 1.0985 0.9004 1.0507	9.284 10.246 12.064 8.900 10.269	3.6871 3.7780 4.5923 3.5829 3.8663	4.6258 4.9956 5.7944 4.4567 5.2074	0.0123 0.0141 0.0146 0.0133 0.0137	0.0049 0.0052 0.0056 0.0054 0.0052	0.0061 0.0069 0.0070 0.0067 0.0069
		Mean S.D. N	1.9670 0.2129 5	0.75626 0.07128 5	0.97238 0.09573 5	10.1526 1.2247 5	3.90132 0.40037 5	5.01598 0.52631 5	0.01360 0.00087 5	0.00526 0.00026 5	0.00672 0.00036 5
3	F	25981 25982 25983 29584 29585	1.973 1.705 1.980 1.915 1.971	0.7653 0.6381 0.7517 0.6916 0.7846	0.9440 0.8504 1.0138 0.9415 0.9990	9.001 9.948 9.567 10.944 10.053	3.4915 3.7231 3.6321 3.9523 4.0020	4.3067 4.9616 4.8986 5.3805 5.0953	0.0114 0.0123 0.0150 0.0154 0.0170	0.0044 0.0046 0.0057 0.0056 0.0068	0.0055 0.0061 0.0077 0.0076 0.0086
		Mean S.D. N	1.9088 0.1169 5	0.72626 0.06031 5	0.94974 0.06423 5	9.9026 0.7132 5	3.76020 0.21526 5	4.92854 0.39394 5	0.01422 0.0231 5	0.00542 0.00097 5	0.00710 0.00127 5
4	F	25992 25993 25994 25995	2.358 1.988 1.895 2.178	0.8394 0.7898 0.7529 0.8485	1.4145 0.9571 0.9367 1.0630	12.075 9.218 10.018 10.498	4.2987 3.6623 3.9801 4.0896	7.2436 4.4381 4.9521 5.1235	0.0163 0.0121 0.0150 0.0140	0.0058 0.0048 0.0060 0.0055	0.0098 0.0058 0.0074 0.0068
		Mean S.D. N	2.1048 0.2059 4	0.80765 0.04470 4	1.09283 0.22148 4	10.4523 1.2038 4	4.00768 0.6549 4	5.43933 1.23760 4	0.01435 0.00177 4	0.00553 0.00053 4	0.00745 0.00170 4

 

Day 15 Relative to Start Date
Group	Sex	Animal	---- Spleen ----			---- Thymus ----			---- Thyroid ----
			Abs	/BW	/BR	Abs	/BW	/BR	Abs	/BW	/BR
1	F	25961 25962 25963 25964 29565	0.656 0.533 0.787 0.593 0.622	0.2409 0.2147 0.2527 0.2485 0.2234	0.3369 0.2532 0.3935 0.2134 0.3170	0.395 0.461 0.518 0.441 0.422	0.1451 0.1857 0.1663 0.1848 0.1516	0.2029 0.2190 0.2590 0.2323 0.2151	0.0190 0.0193 0.0254 0.0160 0.0206	0.0070 0.0078 0.0082 0.0067 0.0074	0.0098 0.0092 0.0127 0.0084 0.0105
		Mean S.D. N	0.6382 0.0946 5	0.23604 0.01637 5	0.32260 0.05046 5	0.4474 0.0464 5	0.16670 0.01860 5	0.22566 0.02139 5	0.02006 0.00343 5	0.00742 0.00060 5	0.01012 0.00164 5
2	F	25971 25972 25973 25974 25975	0.616 0.595 0.753 0.526 0.752	0.2446 0.2194 0.2866 0.2118 0.2831	0.3069 0.2901 0.3617 0.3634 0.3813	0.459 0.583 0.695 0.516 0.471	0.1823 0.2150 0.2646 0.2077 0.1773	0.2287 0.2843 0.3338 0.2584 0.2388	0.0199 0.0352 0.0318 0.0141 0.0256	0.0079 0.0130 0.0121 0.0057 0.0096	0.0099 0.0172 0.0153 0.0071 0.0130
		Mean S.D. N	0.6484 0.1007 5	0.24910 0.03484 5	0.32068 0.04941 5	0.5448 0.0970 5	0.20938 0.03481 5	0.26880 0.04208 5	0.02532 0.00859 5	0.00966 0.00300 5	0.01250 0.00407 5
3	F	25981 25982 25983 29584 29585	0.556 0.863 0.662 0.718 0.672	0.2157 0.3230 0.2513 0.2593 0.2675	0.2660 0.4304 0.3390 0.3530 0.3406	0.704 0.680 0.503 0.586 0.579	0.2731 0.2545 0.1910 0.2116 0.2305	0.3368 0.3392 0.2576 0.2881 0.2935	0.0150 0.0213 0.0178 0.0165 0.0195	0.0058 0.0080 0.0068 0.0060 0.0078	0.0072 0.0106 0.0091 0.0081 0.0099
		Mean S.D. N	0.6942 0.1115 5	0.26336 0.03875 5	0.34580 0.05842 5	0.6104* 0.0817 5	0.23214* 0.03277 5	0.30304 0.3474 5	0.01802 0.00247 5	0.00688 0.00101 5	0.00898 0.00136 5
4	F	25992 25993 25994 25995	0.799 0.546 0.856 0.648	0.2844 0.2169 0.3401 0.2524	0.4793 0.2629 0.4231 0.3163	0.702 0.333 0.568 0.508	0.2499 0.1323 0.2257 0.1979	0.4211 0.1603 0.2808 0.2479	0.0162 0.0096 0.0201 0.0208	0.0058 0.0038 0.0080 0.0081	0.0097 0.0046 0.0099 0.0102
		Mean S.D. N	0.7123 0.1414 4	0.27345 0.05229 4	0.37040 0.09852 4	0.5278 0.1531 4	0.20145 0.05076 4	0.27753 0.10839 4	0.01668 0.00513 4	0.00643 0.00205 4	0.00860 0.00267 4

 

Day 15 Relative to Start Date
Group	Sex	Animal	---- Ovaries ----			---- Uterus ----
			Abs	/BW	/BR	Abs	/BW	/BR
1	F	25961 25962 25963 25964 29565	0.0917 0.1097 0.0878 0.1954 0.1604	0.0337 0.0442 0.0282 0.519 0.0576	0.0471 0.0521 0.0439 0.1030 0.0818	0.458 0.404 0.432 0.732 0.589	0.1682 0.1627 0.1387 0.3068 0.2116	0.2352 0.1919 0.2160 0.3857 0.3002
		Mean S.D. N	0.12900 0.04705 5	0.04912 0.02148 5	0.06558 0.02577 5	0.5230 0.1367 5	0.19760 0.06647 5	0.26580 0.07816 5
2	F	25971 25972 25973 25974 25975	0.1555 0.1299 0.1345 0.1676 0.1332	0.0618 0.0479 0.0512 0.0675 0.0502	0.0775 0.0633 0.0646 0.0839 0.0675	0.443 0.703 0.733 0.429 0.652	0.1759 0.2592 0.2790 0.1727 0.2455	0.2207 0.3428 0.3521 0.2148 0.3306
		Mean S.D. N	0.14414 0.01654 5	0.05572 0.00848 5	0.07136 0.00895 5	0.5920 0.1454 5	0.22646 0.04910 5	0.292220 0.06842 5
3	F	25981 25982 25983 29584 29585	0.0737 0.0968 0.0798 0.1288 0.0810	0.0286 0.0362 0.0303 0.0465 0.0322	0.0353 0.0483 0.0409 0.0633 0.0411	0.408 0.461 1.026 0.509 0.641	0.1583 0.1725 0.3895 0.1838 0.2552	0.1952 0.2299 0.5253 0.2502 0.3249
		Mean S.D. N	0.09202 0.02226 5	0.03476 0.00715 5	0.04578 0.01083 5	0.6090 0.2486 5	0.23186 0.09571 5	0.30510 0.13193 5
4	F	25992 25993 25994 25995	0.1280 0.1348 0.1354 0.0786	0.0456 0.0536 0.0538 0.0306	0.0768 0.0649 0.0669 0.0384	0.827 0.531 0.529 0.747	0.2944 0.2110 0.2102 0.2910	0.4961 0.2557 0.2615 0.3646
		Mean S.D. N	0.11920 0.02727 4	0.04590 0.01089 4	0.06175 0.01641 4	0.6585 0.1519 4	0.25165 0.04742 4	0.34448 0.11278 4

* - Significantly different from the control value, p ≤ 0.05

Nominal Dose:   Group 1 – 0 mg/kg/day                      Group 2 – 100 mg/kg/day Group 3 – 300 mg/kg/day Group 4 – 1000 mg/kg/day

Conclusions:

This study evaluated the potential toxicity of Trixene AS when administered daily for fourteen days via oral gavage to male and female Sprague Dawley rats.
Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology findings, and absolute and relative organ weights.
Treatment with Trixene AS at doses up to 1000 mg/kg had no adverse effect on the parameters evaluated. Therefore, the no-observed-adverse-effect level was 1000 mg/kg when Trixene AS was administered for 14 days via oral gavage to male and female Sprague Dawley rats.

Executive summary:

Trixene AS: A Fourteen-Day Repeat Dose Toxicity Study Following Oral Gavage Administration to Male and Female Sprague Dawley Rats

 

The purpose of this study was to determine the potential toxicity of Trixene AS when administered daily for fourteen days via oral gavage to male and female Sprague Dawley rats.

 

Forty (20/sex) Sprague Dawley rats were randomly assigned to four groups (5 animals/sex/group). Animals were administered control article, peanut oil or Trixene AS at 0, 100, 300, or 1000 mg/kg once daily via oral gavage for 14 consecutive days. Animals were subjected to a full gross necropsy on Study Day (SD) 15.

 

Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology findings, and absolute and relative organ weights.

 

Treatment with Trixene AS at doses up to 1000 mg/kg had no adverse effect on mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology, or absolute and relative organ weights.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral - 14 day

The purpose of the study was to determine the potential toxicity of Trixene AS when administered daily for fourteen days via oral gavage to male and female Sprague Dawley rats.

Forty (20/sex) Sprague Dawley rats were randomly assigned to four groups (5 animals/sex/group). Animals were administered control article, peanut oil or Trixene AS at 0, 100, 300, or 1000 mg/kg once daily via oral gavage for 14 consecutive days. Animals were subjected to a full gross necropsy on Study Day (SD) 15.

Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology findings, and absolute and relative organ weights.

Treatment with Trixene AS at doses up to 1000 mg/kg had no adverse effect on mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology, or absolute and relative organ weights.

Repeated dose toxicity: oral - 28 day

The purpose of this study was to determine the potential toxicity of Trixene AS when administered once daily for at least 28 days via oral gavage to male and female Sprague Dawley rats.

Ninety-six (48/sex) Sprague Dawley rats were randomly assigned to four groups (12 animals/sex). Animals were administered control substance (Peanut Oil) or Trixene AS at 100, 300, or 1000 mg/kg once daily via oral gavage for at least 28 days. Females were dosed for two weeks prior to cohabitation, during cohabitation, through gestation, and to at least postnatal day (PND) 12. Animals were subjected to a full gross necropsy on Study Day (SD) 36 (F0 males), or PND 13 or 14 (parturient F0 females and pups). Females that did not litter were subjected to a full gross necropsy on SD 57.

Parameters evaluated during the study for the F0 generation included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, functional observation battery, vaginal cytology, clinical pathology (clinical chemistry, hematology, and coagulation), thyroid hormone (T4) analysis (males only), gross pathology findings, absolute and relative organ weights, and histopathology findings.

 

Treatment with Trixene AS at doses ≥300 mg/kg/day had no effect on the parameters listed above. Test substance-related changes in body weight and food consumption were observed but were not considered adverse at doses ≥100 mg/kg/day. Body weight and food consumption were statistically significantly decreased compared to the control. All groups gained weight and there was no clear dose response; therefore, the effect on body weight and food consumption were not considered adverse.

Test substance-related changes in thyroid hormone levels of F0 male rats were observed. Mean T4 levels of treated groups were significantly lower than the control group in a dose-dependent manner. In the absence of thyroid organ weight and histopathological effects this finding was not considered an adverse response. Furthermore, no other toxicologically significant observations with respect to survival, clinical observations, body weights, food consumption, and ophthalmology were observed. 

Test substance-related changes in clinical pathology parameters were limited to ~2-fold increases in ALTi activities in animals administered 1000 mg/kg/day consistent with minimal hepatocellular injury; and decreases in measures of erythroid mass with increases in reticulocyte counts in males at all doses and females administered ≥300 mg/kg/day consistent with blood loss or erythrocyte injury and a normal erythrocytic regenerative response. No correlating microscopic changes occurred in the liver; however, test substance-related increased erythropoiesis was observed in the spleen of 4 of 5 males and 1 of 5 females administered 1000 mg/kg/day and 3 of 5 females administered 300 mg/kg/day which correlated with the increases in reticulocyte counts. None of these changes were considered adverse, as the effects on erythroid mass were not of a magnitude to affect the overall well-being of the animal.

The only other test substance-related microscopic change occurred in the testes of 3 of 5 males administered 1000 mg/kg/day and consisted of spermatid retention in some, but not all, Stage IX tubules. Notably, spermiation occurred normally within most tubules and no increase in cellular debris or hypospermia was observed in the epididymides of affected animals. Spermatid retention was considered an adverse change due to the potential effects on reproduction.

In summary, Trixene AS induced repeat dose toxicity in male and female rats at >300 mg/kg/day. The repeat dose no-observed-adverse-effect level (NOAEL) for male rats was determined to be 300 mg/kg/day due to microscopic findings (spermatid retention). The repeat dose NOAEL for female rats was considered to be 1000 mg/kg/day, the highest dose level tested.

Justification for classification or non-classification

Trixene AS does not require classifying for repeated dose toxicity. Whilst there have been observed effects noted on rats male rats in the 28 day repeated dose study, the concentration at which these occur (> 300 mg/kg bw day) are well above the guidance value ranges indicated in table 3.9.2 and factoring paragraph 3.9.2.9.5. (Category 1: ≤ 30 mg/kg bw/day) and 3.9.3 (Category 2: 10 < C ≤ 300 mg/kg bw day) for classification under Regulation (EC) 1272/2008.