diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

occlusive dressing, extended program of histopathology

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

yes

Remarks:

occlusive dressing, extended program of histopathology

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

EC Number:

603-923-2

Cas Number:

135590-91-9

Molecular formula:

C16H18Cl2N2O4

IUPAC Name:

diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: males 190 ± 2.4 g; females 181 ± 2.6 g
- Housing: in Makrolon cages (Type 3) on soft wood granulate, one animal per cage
- Diet (e.g. ad libitum): Altromin 1324 pelleted rat diet (Altromin GmbH, Lage/ Lippe), ad libitum, except for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum, except for the period in which the animals were kept in diuresis cages
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 25 - 75
- Air changes (per hr): approx. 12 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:

occlusive

Vehicle:

polyethylene glycol

Details on exposure:

TEST SITE
- Area of exposure: dorsal
- % coverage: 10
- Type of wrap if used: occlusive bandage (3-layer cellulose bandage, aluminium foil, Fixomull-Stretch and Elastoplast from Beiersdorf/Hamburg)
- Time intervals for shavings or clipplings: At the start of the study and subsequently at least once weekly with an electric clipper


REMOVAL OF TEST SUBSTANCE
- Washing (if done): with warm water
- Time after start of exposure: 6 hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100, 300, 1000 mg/kg
- Concentration (if solution): 50, 150, 500 mg/ml
- Constant volume or concentration used: yes


VEHICLE
- Justification for use and choice of vehicle (if other than water): handling at dosing
- Amount(s) applied (volume or weight with unit): 2 ml/kg


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, occlusive dressing

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Immediatly after preparation and once more at last day of use for application, the active ingredient contents and homogeneity of all formulations
were examined by an analytical laboratory. According to report of 23 October 1991, the homogeneity and stability of the test substance preparations were guaranteed for a period of 18 days and the actually administered test substance concentrations corresponded in high degree to the nominal concentrations.

Duration of treatment / exposure:

29 days

Frequency of treatment:

6 h/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (additionally 5 males and females in control and high dose recovery groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selection of dosages was based on the results of an acute dermal toxicity study in rats, which yielded a LD50-value of > 4000 mg/kg body weight (Report No. 90.0451).
- Post-exposure recovery period in satellite groups: 15 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: behaviour, general health condition (clinical signs), mortality


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for neurological disturbances, damage to the oral mucosa, and impairment of dental growth.


DERMAL IRRITATION (if dermal study): Yes, based on scores of OECD 404
- Time schedule for examinations: prior to each application


BODY WEIGHT: Yes
- Time schedule for examinations: at start of the study and then twice weekly


FOOD CONSUMPTION: Yes, at the start of the study and then twice weekly (same days as body weight)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data


WATER CONSUMPTION: Yes
- Time schedule for examinations: once weekly for a period of 16 hours


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly for opacity of the refracting media of the eyes
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last application (recovery groups at the end of the 2-week recovery period)
- Anaesthetic used for blood collection: Yes (Nembutal), at retrobulbar venous plexus
- Animals fasted: No data
- How many animals: all (a few parameters only for control and high dose group)
- Parameters examined: erythrocytes, haemoglobin, haematocrit, MCV, MCH, MCHC, leucocytes, thrombocytes, differential blood count, reticulocytes*, Heinz bodies*, coagulation time, thromboplastin time, activated partial thromboplastin time, thrombin time, methaemoglobin* (*only performed for control and high dose group)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last application (recovery groups at the end of the 2-week recovery period): after blood sampling from the retrobulbar venous plexus for haematological examination, the animals were killed by opening of the vena cava cranial is under
Nembutal anaesthesia (50 mg/kg body weight, i.p.) and exsanguinated.
- Animals fasted: No data
- How many animals: all
- Parameters examined: sodium, potassium, inorganic phosphorus, uric acid, total bilirubin, creatinine, serum glucose, urea nitrogen, calcium, chloride, ASAT, ALAT, alkaline phosphatase, gamma-GT, LDH, cholesterol, triglycerides, total lipids, total protein, electrophoresis


URINALYSIS: Yes
- Time schedule for collection of urine: in all main groups during the night (about 16 hours) before day 25 of the study. The urine of the recovery animals was not collected, since no effects were observed in the animals of the main groups.
- Metabolism cages used for collection of urine: Yes, individual diuresis cages
- Animals fasted: Yes
- Parameters examined: appearance, colour, pH value, haemoglobin, protein, glucose, ketone bodies, bilirubin, urobilinogen, nitrite, ascorbic acid, sediment (performed only for control and high dose group), volume of urine


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (skin, orifices, eyes, teeth, oral mucosa and internal organs)
HISTOPATHOLOGY: Yes, of control and high dose animals (heart, uterus/ovaries, lungs, adrenals, liver, pituitary, kidneys, thyroid (both lobes), spleen, bone marrow (femur), brain, sternum, both testes, skin (treated & untreated, approx. 2 x 2 cm), seminal vesicle, prostate gland, special macroscopic abnormalities); slides of treated skin, liver, kidneys, testes and macroscopic lesions were read of animals from intermediate groups

Other examinations:

ORGAN WEIGHTS
The absolute weights of the following organs were determined and the relative weights per 100 g body weight calculated: heart, brain, lungs, testes/ovaries, liver, adrenals, kidneys, pituitary, spleen, thyroid

Statistics:

The following data were evaluated statistically at the level of significance p < 0.05:
body weights at the designated measurement times, haematology parameters (except diff. blood count and methaemoglobin), clinical chemistry parameters (except gamma-GT), urinalysis (pH value, volume), absolute organ weights, relative organ weights.
Evaluation was performed by PHARMA DEVELOPMENT INFORMATIC with the aid of a program package for evaluating toxicological studies. The statistical methods used in each case were given on the computer printouts.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

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):

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

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Behaviour and general health condition remained normal throughout the study in all of the treated groups. There were no indications of any neurological disturbances, impairment of dental growth or changes in the oral mucosa which might have been attributed to administration of the test substance. There were no deaths at any time during the study.

BODY WEIGHT AND WEIGHT GAIN
The test substance had no effect on body weight gains in animals of either sex in any of the treatment groups.

FOOD CONSUMPTION
The test substance had no effect on the food consumption of animals of either sex in any of the treatment groups.

WATER CONSUMPTION
Water consumption was variable. No effects could be observed as a result of treatment with the test substance.

OPHTHALMOSCOPIC EXAMINATION
There were no indications of opacity of the refracting media of the eyes.

HAEMATOLOGY
At the end of the treatment period slight decreases in erythrocyte counts, haemoglobin content and haematocrit value in the females from the highest dose group. This finding might be related to the test substance and indicative of a marginal anaemia. This effect could not be observed at the end of the recovery period. Additional correlates indicative of haematoxicity could not established by clinical biochemistry or pathology. No other changes attributable to the test substance could be detected.

CLINICAL CHEMISTRY
Statistical analysis revealed only a decrease in total bilirubin in the females of the highest dose group at the end of treatment. This effect showed a dose-dependency and was therefore considered to be related to the treatment with the test compound. No other changes were noted at the end of treatment.
Evaluation of the serum electrophoresis data gave no convincing evidence of substance-related changes at the end of treatment. Only a marginal increase in globulines was noted in the females treated at 300 and 1000 mg/kg body weight. However, no biological significance could be assigned to this change.

URINALYSIS
The urinary status of all animals remained unaffected by dermal treatment with the test substance. There was no evidence of any damage to the renal system. The urine of the large majority of animals in all groups was clear and yellow in colour. At the end of the treatment period, the group mean pH values varied between 6.7 and 6.9 in the males and between 6.2 and 6.8 in the females. The group mean urinary volume varied between 6.1 and 7.4 ml in the males and between 2.8 and 4.3 ml/16h in the females. Examination of the urine for protein, glucose, haemoglobin, bilirubin, urobilinogen, ascorbic acid, nitrite and ketone bodies gave no indication of substance-related changes; the same applies to the examination of urinary sediments in the
control and high-dose animals at the end of the treatment period.

ORGAN WEIGHTS
Statistical analysis of the absolute and relative organ weights indicated no changes in any of the treated groups as compared with the control group.

GROSS PATHOLOGY
Neither topically nor systemically any adverse reaction attributable to the test compound administered was to be found at morphological investigation of skin and internal organs.

HISTOPATHOLOGY: NON-NEOPLASTIC
Neither topically nor systemically any adverse reaction attributable to the test compound administered was to be found at microscopical investigation of skin and internal organs.

OTHER FINDINGS
The test substance proved to be non-irrating to the skin and histopathology indicated also no changes attributable to the test compound.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

2

Key result

Dose descriptor:

LOAEL

Remarks:

sytemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

Haematology parameters:

	Dose level (mg/kg bw/d)
	males				females
Parameter	0	100	300	1000	0	100	300	1000
Erythrocytes (10e12/L)	6.65	6.79	7.08	6.76	6.85	6.32	6.64	6.10*
Hb (g/L)	131	137	139	137	131	130	128	125
Haematocrit	0.40	0.41	0.42	0.41	0.42	0.40	0.40	0.38
MCH (10e-12g)	20	20	20	20	19	21*	19	21*
MCHC (g/L)	327	333	331	336	314	327*	321	330*

* p<= 0.05

Conclusion:

Based on the results of this repeated-dose dermal toxicity study (21 treatments in 29 days) with Hoe 107892 00 ZC94 0001 in the Wistar rat, the NO OBSERVABLE ADVERSE EFFECT LEVEL (NOAEL) for systemic toxicity is considered to be 300 mg/kg bw/day.

Repeated dermal treatment with 1000 mg/kg body weight caused a marginal anaemia and a decrease in total bilirubin in the female rat.

Under the conditions of this study, the test substance in the form of a suspension in PEG 400 proved to be non-irritating to the treated skin.