6-chloro-5-(2-chloroethyl)-1,3-dihydroindol-2-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 1995-January 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD and in accordance with GLP. The study material is well characterize

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Principles of method if other than guideline:

The method followed was that outlined in Annex to Directive 92/69/EEC, Part B, Method B7, and OECD guideline for testing of chemicals No.407 "Repeated Dose Oral Toxicity- Rodent: 28 day or 14 day study".
The study was run in two stages. Due to a clerical error in the protocol regarding the high dosage necessitating termination Group 4, the study was run in two stages. The first stage consisted of the intermediate, low dosage and control groups. The second stage consisted of the high dosage group and concurrent control

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

6-chloro-5-(2-chloroethyl)-1,3-dihydroindol-2-one

EC Number:

421-320-0

EC Name:

6-chloro-5-(2-chloroethyl)-1,3-dihydroindol-2-one

Cas Number:

118289-55-7

Molecular formula:

Hill formula: C10 H9 Cl2 N O CAS formula: C10 H9 Cl2 N O

IUPAC Name:

6-chloro-5-(2-chloroethyl)-2,3-dihydro-1H-indol-2-one

Test material form:

solid: crystalline

Details on test material:

Expiry date: September 1996
Purity: >99%
Date received: 14 September 1995
Pharmaceutical intermediate,

Test animals

Species:

rat

Strain:

other: Sprague-Dawley origin (Crl:CD BR VAF PLUS™)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent, England
- Age at study initiation: 28 ± 1 days old,
- Weight at study initiation: in a weight range of 69 to 87 g on arrival
- Fasting period before study: Food was withdrawn overnight prior to collection of samples
- Housing: All rats were initially caged, as far as possible, in groups of five according to sex in metal cages with wire mesh floors
- Diet (e.g. ad libitum): standard pelleted laboratory rodent diet
- Water (e.g. ad libitum): tap water
- Acclimation period: 5 days before

ENVIRONMENTAL CONDITIONS
- Temperature (°C): range 17 to 22
- Humidity (%): 36 to 59
- Air changes (per hr): approximately 19
- Photoperiod (hrs dark / hrs light):12artificial light (0700-1900 hours) in each 24- hour period.

IN-LIFE DATES: From: Sept 8th, 1993 To: Sept. 28th, 1993

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1 % methyl cellulose aqueous solutioncontaining 1% Tween 80

Details on oral exposure:

The high dosage was selected on the basis of available toxicity data and a preliminary oral toxicity investigation performed at Huntingdon Life Sciences schedule number PFZ/750. intermediate and low dosage levels were selected on the basis of the key dosages relative to EEC labelling requirements

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

dose level:Control, 15, 150 mg/kg other group had a control and then 300mg/kg dosing groups.

Duration of treatment / exposure:

concentration: Groups 2, 3 and 6 using a syringe and rubber catheter at a dose volume of 10 ml/kg/day.
Control animals received the vehicle at the same dose volume

Frequency of treatment:

Each animal received a constant dosage based on its most recently recorded bodyweight. Animals were treated once daily for at least twenty-eight consecutive days.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
300 mg/kg
Basis:
other: by gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
150 mg/kg
Basis:
other: by gavage

Doses / concentrations 3

Remarks:

Doses / Concentrations:
15 mg/kg
Basis:
other: by gavage

No. of animals per sex per dose:

Groups of ten rats (five males and five females)

Control animals:

yes

Details on study design:

Prior to dosing, the test substance formulations for Groups 2, 3 and 6 were mixed by inversion. Subsequent mixing using a magnetic stirrer occurred for a period of at least 10 minutes before dosing commenced.Blood was withdrawn under light ether anaesthesia from the orbital sinus of all rats prior to termination (Week 4). Further removal of blood samples for re-analysis was carried out for individual animals, on Day 28. After 28 days of treatment (Day 29) all animals surviving treatment were killed by carbon dioxide asphyxiation and a complete autopsy undertaken.Specified organs were weighed and relevant tissue samples were fixed for microscopic examination

Positive control:

N/A

Examinations

Sacrifice and pathology:

After 28 days of treatment (Day 29) all animals surviving treatment were killed by carbon dioxide asphyxiation and a complete autopsy undertaken.Specified organs were weighed and relevant tissue samples were fixed for microscopic examination

Statistics:

If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75% ), the proportion of values different from the mode were analysed by Fisher's exact test (1) followed by Mantel's test for a trend in proportions (2). Otherwise:

Bartlett's test (3) was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1% level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.

If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out followed by Williams' test (5) for a dose related response.

If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks (4) was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test,. (6)).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two female animals (579 & 589) treated with 300 mg/kg/day : killed on humane grounds on Days 16 and 20 respectively, Clinical signs prior to sacrifice were piloerection, thin appearance, pallor of extremities, hunched posture, lethargy, increased salivation and swollen muzzle There were no clinical signs for rats treated at 15 or 150 mg/kg/day throughout the treatment period. Fur loss on shoulders was recorded for two male rats (47 o & 49o) treated at 300 mg/kg/day from Day 22 to termination. This finding often accompanies increased salivation and can be a result of fur getting wet. Alternatively, fur loss may have been due to fighting and is not considered to be of toxicological importance piloerection on D1 ( 1F/1)

Mortality:

mortality observed, treatment-related

Description (incidence):

Two female animals (579 & 589) treated with 300 mg/kg/day : killed on humane grounds on Days 16 and 20 respectively, Clinical signs prior to sacrifice were piloerection, thin appearance, pallor of extremities, hunched posture, lethargy, increased salivation and swollen muzzle There were no clinical signs for rats treated at 15 or 150 mg/kg/day throughout the treatment period. Fur loss on shoulders was recorded for two male rats (47 o & 49o) treated at 300 mg/kg/day from Day 22 to termination. This finding often accompanies increased salivation and can be a result of fur getting wet. Alternatively, fur loss may have been due to fighting and is not considered to be of toxicological importance piloerection on D1 ( 1F/1)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences from control for bodyweight gain for rats of the intermediate and low dosage groups.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In higher group lower packed cell volume, haemoglobin concentration, and red blood cell counts with subsequent marginal disturbances in the calcuated indicexes but .there was no evidence of hemolysis or blood loss and aetiology questionable.

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

spleen slightly lower and liver slightly higher than control- males- high dose group

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examinations performed at terminal kill revealed caecal distension in 4/5 male and 2/3 female rats treated at 300 mg/kg/day and in 2/5 male rats treated with 150 mg/kg/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examinations performed at terminal kill revealed caecal distension, at high and mid doses, Increased pelvic dilatation in the kidney, reduction in adipose tissue and a small thymus at high dose.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A minor immunosuppressive effect, chiefly manifested in the thymus and splenic peri-arteriolar lymphoid sheath, was noted at the high dosage level. In the kidneys some inflammatory and hyperplastic changes of the renal pelvis were seen in the high dose.

Details on results:

In a small number of animals receiving 300 mg/kg/ day there was a varying degree of minor renal pelvic dilatation as well as occasional pelvic inflammation/pyelitis. In two female animals there was also an associated hyperplasia of the pelvic transitional epithelium

The following comments are made in summary: Mortality
Two female rats from the high dosage of 300 mg/kg were killed on humane grounds on Days 16 and
20. Clinical signs prior to sacrifice were piloerection, thin, pallor of extremities, hunched posture, lethargy, increased salivation and swollen muzzle.

The macroscopic examination of rat no. 57 revealed crater like depressions, thickened and white forestomach, congested lungs, brown stained fur of the cranial region and forepaws, minimal adipose tissue, a raised subcapsular area on the spleen and a small uterus. The microscopic examination concluded that ulceration of the non-glandular epithelium of the stomach was the factor contributing to the death of the animal.

Macroscopic examination of rat 58 revealed moist fur in the perioral region, pale thyroids, oedematous thymus, minimal adipose tissue and pale kidneys. The microscopic examination concluded that pyelitis, tubular basophilia and inflammatory exudate in the pelvic cavity of the kidneys were the factors contributing to the death of this animal.

Bodyweight

Bodyweight gains were slightly lower than control for male rats treated at 300 mg/kg/day. Haematology
For high dosage group male and female rats higher platelet counts and lower neutrophil, lymphocyte,
eosinophil, basophil and white blood cell counts were recorded. Neutrophil, lymphocyte and white blood cell counts were also lower than control for rats of the intermediate dosage group.

Lower than control packed cell volume (PCV), haemoglobin concentration (Hb) and red blood cell counts (RBC) and consequent marginal disturbances in the calculated indices were recorded for rats receiving 300 mg/kg/day. PCV, Hb and RBC were also lower than control and MCV and MCH higher than control for rats at 150 mg/kg/day.

Biochemistry

Higher than control glutamic-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) levels were recorded for male rats of the high dosage group. Urea nitrogen and glucose levels were higher than control for male rats treated at 300 mg/kg/day.

Organ weights

Spleen weights (absolute) were lower than control and liver weights (bodyweight adjusted) were higher than control for male rats of the high dosage group.

Macroscopic pathology

For rats of the high dosage group increased pelvic dilatation (males only), small thymus (males and females) and a reduction in adipose tissue (males and females) were observed.

Caecal distention was recorded in male and female rats of the high dosage group and among male rats from the intermediate dosage group.

Microscopic pathology

A minor immunosuppressive effect, chiefly manifested in the thymus and splenic peri-arteriolar lymphoid sheath, was noted at the high dosage level. In the kidneys some inflammatory and hyperplastic changes of the renal pelvis were also seen in the high dosage group.

Other findings

There were no other differences from control for the remaining parameters measured, including clinical signs, food consumption and water consumption, that were considered to be related to treatment.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Concluded to be no-observed-adverse-effect level (NOAEL) for CP-89 ,575 in the rat.

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No-observed-effect level (NOEL) for CP-89,575 in the rat when administered orally for 28 days.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Immune system: A slight increase in the paracortical cellularity in the cervical lymph nodes of occasional animals may be associated with these disturbances to the immune system.

Dosagelevel(mg/kg/day)	0	15	Males 150	0	300	0	15	Females 150 0		300
Spleen
ReducedcellularityofPALS	0	0	0	0	3	0	0	0	0	3
Thymus
Involution	0	0	0	0	4	0	0	0	0	2
Cervicallymphnode
Increased cellularity (generalised)	3	1	1	5	1	1	2	1	4	2
Reduced cellularity (generalised)	0	0	0	0	0	0	0	0	0	2
Increasedcellularity-paracortex	0	0		0	1	0	0	1	0	0

Applicant's summary and conclusion

Conclusions:

Target organs for toxicity of the high dosage level of 300 mg/kg/day were the immune system and kidney
intermediate dosage of 150 mg/kg/day : concluded to be no-observed-adverse-effect level (NOAEL) for CP-89 ,575 in the rat.
15 mg/kg/day: no-observed-effect level (NOEL) for CP-89,575 in the rat when administered orally for 28 days