Ethanone, 2,2-dichloro-1-(2,3-dihydro-3-methyl-4H-1,4-benzoxazin-4-yl)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 March 1990 to 6 March 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2-dichloro-1-[(3R)-3-methyl-3,4-dihydro-2H-1,4-benzoxazin-4-yl]ethan-1-one; 2,2-dichloro-1-[(3S)-3-methyl-3,4-dihydro-2H-1,4-benzoxazin-4-yl]ethan-1-one

EC Number:

619-372-6

Cas Number:

98730-04-2

Molecular formula:

C11H11Cl2NO2

IUPAC Name:

2,2-dichloro-1-[(3R)-3-methyl-3,4-dihydro-2H-1,4-benzoxazin-4-yl]ethan-1-one; 2,2-dichloro-1-[(3S)-3-methyl-3,4-dihydro-2H-1,4-benzoxazin-4-yl]ethan-1-one

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products Inc., Denver, Pennsylvania 17517, USA
- Age at study initiation: 6-7 months
- Weight at study initiation: 6.35-9.80 kg (males); 5.45-7.75 kg (females)
- Fasting period before study: none
- Housing: individually during the working day and in pairs of same sex and group overnight (after removal of food)
- Diet: 400 g/dog/day of SQC Laboratory Diet A, Expanded, pellets (Special Diets Services Ltd., Witham, UK) ad libitum (except overnight prior to blood collection and necropsy)
- Water: filtered mains drinking water ad libitum
- Acclimation period: minimum of 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 16-22°C
- Humidity: 40-80%
- Air changes: minimum of 10/hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 1 March 1990 To: 6 March 1991

Administration / exposure

Route of administration:

oral: capsule

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Calculations of the required amount of GCA154281 to be dosed were based on each dog's weight from the previous week. The correct amount of test substance was weighed and placed into empty gelatin capsules. Capsules were prepared weekly for each individual dog and prior to dosing were stored in a sealed container at ambient temperature.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 1, 5, 40, 80 mg/kg/day
Basis:
other: nominal

No. of animals per sex per dose:

4

Control animals:

other: yes: empty capsule only

Details on study design:

- Dose selection rationale: based on results from a 28 day oral capsule administration toxicity study in the beagle dog where dose levels of 80, 100, 200 and 300 mg/kg/day were dosed and 80 mg/kg/day was the maximum tolerated dose in this pilot study.

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily before feeding. Additional observations were made throughout the working day as necessary.

DETAILED PHYSICAL EXAMINATION: Yes
- Time schedule: Pre-dose and during weeks 12, 25 and 52

AUDITORY RESPONSE: Yes
- Time schedule: pre-dose and in weeks 13, 26 and 52

BODY WEIGHT: Yes
- Time schedule: weekly throughout the study

FOOD CONSUMPTION: Yes
- Time schedule: daily

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: prior to treatment and during weeks 13, 26 and 52
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to treatment and during weeks 13/14/15, 26 and 52. Samples were collected prior to dosing.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: haemoglobin, mean cell volume, erythrocyte count and indices (mean cell haemoglobin, mean cell haemoglobin concentration, packed cell volume), total and differential white blood cell count, platelet count, Heinz bodies, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to treatment and during weeks 13, 26 and 52. Samples were collected prior to dosing.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase, creatine phosphokinase, sodium, potassium, chloride, inorganic phosphorus, total protein, total lipids, albumin, globulin, A/G ratio, calcium, total bilirubin, urea, creatinine, glucose, total cholesterol.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to treatment and during weeks 13, 26 and 52 by direct catheterisation of the bladder. Samples were collected prior to dosing.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters examined: specific gravity, protein, ketones, blood, reducing substances, pH, glucose, total bilirubin, urobilinogen, colour, turbidity, microscopy of sediment

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (all dogs given a complete necropsy)
ORGAN WEIGHTS: Yes
- Organs weighed: adrenals, heart, liver, pituitary, spleen, brain, kidneys, ovaries, testes with epididymes, thyroids.
HISTOPATHOLOGY: Yes (all animals)
- Tissues/organs: all gross lesions, brain (with brainstem), gallbladder, adrenals, aorta, rib and bone marrow, caecum, colon, duodenum, epididymes, eyes (with optic nerve), oesophagus, heart, ileum, jejunum, kidneys, liver, lungs (with mainstem bronchi), lymph nodes (mandibular, popliteal and mesenteric), ovaries, pancreas, parathyroid glands, prostate, sciatic nerve, pituitary, rectum, salivary glands (submaxillary), spinal cord (lumbar, cervical, thoracic), spleen, sternum (with bone marrow), skeletal muscle (Quadriceps), stomach, testes, epididymes, thymus, thyroids (with parathyroids), tongue, trachea, urinary bladder, skin and mammary gland, uterus and vagina.

Statistics:

All variables apart from organ weights were analysed using 2-way analysis of variance (ANOVA). Pairwise comparisons for each sex separately were made using Dunnett's test apart from pre-dose variables for which pair-wise comparisons were made using a protected t-test. Levene's test for equality of variances across groups, between sexes and for any interaction were also performed. When these tests showed evidence of group effects or a sex-group interaction, the data were re-analysed using non-parametric methods for each sex separately. These were Kruskal-Wallis (ANOVA), Wilcoxin rank sum test (pair-wise comparisons) and Terpstra-Jonckheere (dose response) test, where appropriate. If Levene's test showed evidence of differing variances between the sexes only then a one-way ANOVA, regression (if applicable) and Dunnett's (or t-tests) were performed for each sex separately. Organ weights were analysed using analysis of covariance (ANCOVA) using the necropsy bodyweight as a covariate.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No treatment-related effects

BODY WEIGHT AND WEIGHT GAIN: No toxicologically significant effects. Body weight gain at 40 and 80 mg/kg/day males was lower than male controls at times during the study. The differences were only statistically significant at 80 mg/kg/day for the period weeks 13 - 26.

FOOD CONSUMPTION: No treatment-related effects.

OPHTHALMOSCOPIC EXAMINATION: No treatment-related effects.

AUDITORY RESPONSE: No treatment-related effects.

HAEMATOLOGY: Haemoglobin, red cell count and packed cell volume were lower than controls for 80 mg/kg/day males. Haemoglobin and packed cell volume were also reduced for 40 mg/kg/day males at week 52 and mean cell volume and mean cell haemoglobin for 80 mg/kg/day males were reduced at week 52.

CLINICAL CHEMISTRY: Creatine phosphokinase activities were lower for male treated groups during weeks 13 and 26 and were reduced for female treated groups at 40 and 80 mg/kg/day in week 52. Creatinine levels were lower than controls for males at 40 and 80 mg/kg/day during weeks 13, 26 and 52 and for females at 40 and 80 mg/kg/day in week 52. Bilirubin was increased compared to controls for 80 mg/kg/day males and females in week 13 and for 40 mg/kg/day females and 80 mg/kg/day males and females in weeks 26 and 52.

URINALYSIS: No treatment-related effects

ORGAN WEIGHTS: Absolute and adjusted kidney and liver weights at 40 and 80 mg/kg/day (males and females) were generally higher than controls.

GROSS PATHOLOGY: No treatment-related effects

HISTOPATHOLOGY: 80 mg/kg/day males showed a slight increase compared to controls in the deposition of pigment in kidney proximal tubular cells.

Effect levels

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Reduced body weight gain, minor disturbances of clinical pathology parameters and increases in liver and kidney weights with pigment deposition in the kidney at 40 and/or 80 mg/kg/day

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Intergroup comparison of body weight change (kg) – males

weeks	Dose level of CGA154281 (mg/kg/day)
	0	1	5	40	80
-1 to 13	1.79	2.21	1.62	1.61	1.26
13 to 26	0.69	0.55	0.80	0.36	0.06*DR
26 to 39	0.28	0.16	0.23	-0.01	0.38
39 to 52	-0.04	0.04	-0.05	-0.23	-0.06
* Statistically significant difference from control group mean, p<0.05 DR + significant using the dose-response test

 

Table 2: Intergroup comparison of selected haematology parameters

 

Parameter		Dose level of CGA154281 (mg/kg/day)
		males					females
		0	1	5	40	80	0	1	5	40	80
haemoglobin	wk 13	16.5	16.3	15.0	15.4	14.9*DR	16.3	15.1	16.2	15.4	15.1
	wk 26	16.8	17.2	16.0	15.8	14.6*DR	17.1	14.7	16.6	15.3	15.4
	wk 52	17.6	17.7	16.7	15.6*	15.1**	17.5	16.4	17.4	16.4	15.8
red cell count	wk 13	7.05	7.12	6.34	6.68	6.41*DR	6.93	6.55	6.93	6.59	6.43
	wk 26	7.10	7.32	6.63	6.77	6.35*DR	7.14	6.16	7.01	6.45	6.51
	wk 52	7.25	7.43	6.79	6.58	6.52**DR	7.21	6.90	7.14	6.92	6.64
packed cell volume	wk 13	49.0	48.7	44.4	45.4	44.0*DR	48.2	44.8	47.8	45.7	44.7
	wk 26	49.2	49.9	46.9	45.7	42.8**DR	49.6	42.3	48.7	44.8	45.0
	wk 52	49.9	50.3	47.2	44.4*	43.1**	49.3	46.6	49.3	46.9	45.1
mean cell volume	wk 13	69.5	68.4	70.2	68.1	68.6	69.5	68.5	69.0	69.4	69.5
	wk 26	69.2	68.2	70.7	67.5	67.3	69.5	68.7	69.7	69.5	69.1
	wk 52	68.9	67.7	69.8	67.5	66.2*DR	68.4	67.6	69.0	67.9	68.0
mean cell haemoglobin	wk 13	23.5	22.9	23.7	23.1	23.2	23.4	23.1	23.3	23.4	23.5
	wk 26	23.6	23.5	24.1	23.4	23.0	24.0	23.9	23.8	23.8	23.7
	wk 52	24.3	23.9	24.6	23.7	23.2*DR	24.3	23.8	24.4	23.7	23.8
* Statistically significant difference from control group mean, p<0.05 ** Statistically significant difference from control group mean, p<0.01 DR significant using the dose-response test

 

Table 3: Intergroup comparison of selected clinical chemistry (blood) parameters

 

Parameter		Dose level of CGA154281 (mg/kg/day)
		males					females
		0	1	5	40	80	0	1	5	40	80
creatine phospho-kinase	wk 13	199	136	134	135	108*DR	153	127	194	132	169
	wk 26	227	150***	146***	156***	140***	182	149	165	152	146
	wk 52	137	136	119	173	147	199	163	157	123	126*DR
creatinine	wk 13	81	78	79	71	71*DR	77	76	76	72	72
	wk 26	96	88	84	75**	80*	81	87	82	75	80
	wk 52	86	83	79	69	77*DR	84	100	79	71	70*DR
bilirubin	wk 13	2.0	1.8	1.9	1.9	2.6*DR	2.4	1.8	2.1	2.3	2.9*DR
	wk 26	2.9	2.6	2.6	2.8	3.4	2.8	2.4	2.6	3.2	3.7*
	wk 52	2.4	2.5	2.7	2.7	3.2*	2.8	2.6	2.7	3.6	3.5**DR
* Statistically significant difference from control group mean, p<0.05 ** Statistically significant difference from control group mean, p<0.01 *** Statistically significant difference from control group mean, p<0.001 DR significant using the dose-response test

 

 

Table 4: Intergroup comparison of selected organ weights adjusted to final bodyweight

 

Organ	Dose level of CGA154281 (mg/kg/day)
	males					females
	0	1	5	40	80	0	1	5	40	80
Liver	296.9	327.5	304.0	421.3**	392.0*	249.8	250.3	280.8	354.2*	325.3
Kidney	50.408	52.299	56.188	61.400**	60.749	37.457	43.124	38.352	44.443	43.442
* Statistically significant difference from control group mean, p<0.05 ** Statistically significant difference from control group mean, p<0.01

 

Table 5: Intergroup comparison of selected histopathological findings in kidney – males

 

Finding	Dose level of CGA154281 (mg/kg/day)
	0	1	5	40	80
pigment	1	2	1	2	4

 

Applicant's summary and conclusion

Conclusions:

Oral administration of benoxacor tech to the dog for 52 weeks at dose levels up to 80 mg/kg/day was well tolerated.
The NOEL to male and female dogs was 5 mg/kg/day.

Executive summary:

Benoxacor tech was dosed to groups of 4 male and 4 female beagle dogs (oral administration by capsule) for 52 weeks at dose levels of 0 (control), 1, 5, 40 and 80 mg/kg/day.

Oral administration of benoxacor tech to the dog for 52 weeks at dose levels up to 80 mg/kg/day was well tolerated. Changes associated with treatment included a reduced body weight gain, minor disturbances of blood clinical pathology parameters and increases in liver and kidney weights with pigment deposition in the kidney at dose levels of 40 and/or 80 mg/kg/day.

The NOEL to male and female dogs was 5 mg/kg/day.