Nitrapyrin

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 April 1991 to 13 February 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to valid guidelines, and the study was conducted under GLP conditions.

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:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Directive 67/548/EEC, Page 127,1984

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MAFF, Page 33, 1985

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nitrapyrin

EC Number:

217-682-2

EC Name:

Nitrapyrin

Cas Number:

1929-82-4

Molecular formula:

C6H3Cl4N

IUPAC Name:

2-chloro-6-(trichloromethyl)pyridine

Test material form:

solid: crystalline

Details on test material:

- Appearance: white crystals

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 5 - 6 months
- Weight at study initiation: 2980 - 3888 g (males); 3099 - 3528 g (females)
- Housing: individually in stainless steel cages in rooms designed to maintain adequate environmental conditions
- Diet: rabbits were provided 2 oz (pre-study), 4 oz/day diet
- Water: ad libitum
- Acclimation period: at least 14 days

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: test material was applied to a 10 x 15 cm, clipped area, on the back of each rabbit
- % coverage: approximately 10 %
- Type of wrap if used: test material was covered with an absorbent gauze patch. The patch was backed by non-absorbent cotton and was held in place with an elastic jacket. The gauze patch was moistened with distilled water prior to applying the solid test material to aid in maintaining it at the application site.

REMOVAL OF TEST MATERIAL
Jackets and patches were removed approximately 6 hours after application and the treated area was wiped with a water-dampened disposable towel to remove any residual test material. Animals were acclimated to jackets prior to the beginning of the dosing period.

TEST MATERIAL
The test material was ball-milled into a fine powder before use. Neat test material was used to dose animals, the amount being applied/ animal was adjusted weekly based upon the individual animal's most recent body weight data.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

- Range-finder: 4 days
- Main test: 5 days/week, 15 applications, excluding weekends and holidays, for up to 22 days. Rabbits were dosed for at least two days immediately prior to necropsy.

Frequency of treatment:

- Range-finder: Once daily
- Main test: Once daily, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Range finder

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Main test

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Main test

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Main test

No. of animals per sex per dose:

- Range-finder: 2 animals/sex/dose
- Main test: 5 rabbits/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale: The 1000 mg/kg/ day dose level (selected for the range-finder) represented a limit test as outlined in the EPA Testing Guidelines (EPA-FIFRA, 1984). For the main test, the high dosage was based on the results of the range-finder which did not produce clear evidence of toxicity. The remaining dose levels were expected to provide dose-response data for any potential toxicity observed in high-dose group animals and to ensure definition of a no-observed-effect dose level of the test material.
- Rationale for animal assignment (if not random): Animals were randomly assigned to control and treatment groups using a randomisation program based upon body weights.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations included: twice daily visual evaluation for morbidity and mortality and the availability of feed and water; once daily visual evaluation for alterations in skin, fur, mucous membranes, respiration, central nervous system function and animal behaviour.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- Observations included: Detailed evaluation of skin, fur, mucous membranes, respiration, central nervous system function (including tremors, convulsions and diarrhoea), swelling, and animal behaviour.

DERMAL IRRITATION: Yes
- Time schedule for examinations: on removal of wrap daily in the range-finding test and on the last day of doing each week and the day before necropsy in the main test (grading scheme outlined in Table 1).

BODY WEIGHT: Yes
- Time schedule for examinations: All rabbits were weighed during the pre-study period and weekly during the dosing periods.

FOOD CONSUMPTION: No
- Feed consumption was not quantitated since the rabbits typically consume their entire ration of 2 or 4 oz/ day.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all rabbits were examined by a veterinarian pre-study and at necropsy.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood was collected the day prior to necropsy via puncture of the ear vein on test day 22. Samples were mixed with EDTA and blood smears were prepared and stained with Wright's Stain.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All animals
- Parameters checked included: haematocrit, haemoglobin concentration, erythrocyte count, total leukocyte count, platelet count, differential counts of 100 leukocytes, and erythrocyte, leukocyte and platelet morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood was collected the day prior to necropsy via puncture of the ear vein on test day 22. Samples were held on ice and serum harvested.
- Animals fasted: No
- How many animals: All animals
- Parameters checked included: alkaline phosphatase, alanine aminotransferase, urea nitrogen, creatinine, total protein, albumin, globulin, glucose, total bilirubin and electrolytes (Na, K, P, Cl and Ca).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

TERMINAL PROCEDURES
The animals were anaesthetised with carbon dioxide and euthanatised by decapitation on test day 23. Tracheas were clamped and the rabbits exsanguinated.
Terminal body weights were recorded for all rabbits.

GROSS PATHOLOGY: Yes
A complete gross examination of tissues was conducted on all animals by a Veterinary Pathologist. The nasal cavity was flushed and the lungs inflated to an approximately normal inspiratory volume with neutral, phosphate-buffered 10 % formalin.
Representative samples of tissues listed below were collected and preserved in formalin. Samples of skin were obtained from: 1) the clipped test material application site; 2) an unclipped site adjacent and caudal to the application site (control tissue for possible mechanical irritation of skin by wrapping materials and/or during clipping); and, 3) (for comparison purposes between treated and untreated skin) the mammary region to include the mammary gland.

Tissues collected and preserved at necropsy included: adrenals, aorta, appendix, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, colon, duodenum, epididymides, eyes, gallbladder, gross lesions, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mammary gland, mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues, oesophagus, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve, pituitary, prostate, rectum, sacculus rotundus, salivary glands, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina.

HISTOPATHOLOGY: Yes
Tissues from the liver, kidneys, treated and untreated skin from all animals were processed. Tissues were sectioned at approximately 6 µm thick, stained with haematoxylin and eosin and examined using light microscopy by a Veterinary Pathologist. A semi-quantitative histologic grading system was used in estimating the degree of epidermal hyperplasia and inflammation of the skin. The number of cells from the basal layer to the stratum corneum of the epidermis was counted to grade the degree of epidermal hyperplasia. Up to four cells thick was considered to be within normal limits, 5 to 7 cells thick was graded as very slight, 8 to 10 cells thick was slight and 11 to 15 cells thick was moderate. The number of white blood cells per 400x microscopic field was counted to grade the degree of dermal inflammation. Up to 10 was considered to be within normal limits, 11 to 20 was graded as very slight, 21 to 30 was graded as slight and 31 to 50 was graded as moderate.

Statistics:

All parameters examined statistically were first tested for equality of variance using Bartlett's test. If the results from Bartlett's test were significant, then the data for the parameter were subjected to a transformation to obtain equality of the variances. The transformations that were examined were the common log, the inverse, and the square root, in that order with a Bartlett's test following each transformation. When Bartlett's test was satisfied no further transformations were applied, or, if none of the transformations result in homogeneous variances the transformed data or raw data with the lowest Bartlett's statistic was used. The selected form of the data was then subjected to the appropriate parametric analysis.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

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):

See "Details on results" for information

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no treatment-related observations during the study suggestive of systemic toxicity.

SKIN REACTIONS
Localised signs of reaction to treatment consisted of dermal irritation at the test site of various animals in all male treatment groups and females from the 500 and 1000 mg/kg/day groups. Dermal irritation was characterised by very slight (barely perceptible) to well defined erythema and/or very slight oedema. One male and two females in the 500 mg/kg/ day group had slight to moderate scaling at the site of application.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related effects on mean body weights were noted in male or female rabbits at dose levels up to 1000 mg/kg/day.

HAEMATOLOGY
No treatment-related changes in haematologic parameters were noted between control and treated rabbits administered test material.

CLINICAL CHEMISTRY
No treatment-related changes in clinical chemistry parameters were noted between control and treated rabbits administered test material.

ORGAN WEIGHTS
No significant effects were noted in the organ and organ to body weight ratios in any dose group. However, statistically significant increases in absolute and relative liver weights were observed in the 1000 mg/kg/ day male and female rabbits. The minor effect on the liver weight, the absence of gross or histopathologic findings, as well as normal values for clinical chemistry parameters which would indicate liver toxicity, all indicate that this observation was not toxicologically significant.

GROSS PATHOLOGY
Gross pathologic observations attributable to treatment were confined to the dermal test site. Three of five 1000 mg/kg/ day group males had erythema at the dermal test site. Female observations consisted of erythema and/or scales in four of five 500 mg/kg/day dose animals and three of five 1000 mg/kg/day animals. The only other gross pathologic finding was focal aplasia of the right horn of the uterus in one 500 mg/kg/ day rabbit which was considered not treatment-related.

HISTOPATHOLOGY
Histopathologic evaluation at the site of topical application of the test material was based upon the comparison of sections of skin collected from the dermal test site and that adjacent to the test site. Further comparison was made between the skin sections obtained from the control rabbits and rabbits treated with the test material. Histopathologic changes at the dermal test site were present in all treatment groups, 100, 500 and 1000 mg/kg/ day. These were characterised by very slight to moderate epidermal hyperplasia, focal to multifocal epidermal pustules, multifocal to diffuse hyperkeratosis, multifocal parakeratosis, very slight to moderate dermal inflammation, and multifocal dermal oedema. In males, there generally was a dose-related increase in the incidence and/or severity of effects in the skin at all dose levels. In females there was a dose-related increase in the incidence and/or severity of skin changes when comparing the 100 and 500 mg/kg/ day animals, whereas, the effects in the 500 and 1000 mg/kg/ day females were similar.
The occasional observations of multifocal hyperkeratosis, very slight epidermal hyperplasia and very slight to slight dermal inflammation, observed in the untreated skin adjacent to the dermal test site in some control and treated animals were considered to be associated with friction caused by the wrapping and unwrapping of the jackets used to hold the test material in place.
Histopathologic evaluation of the liver and kidneys from all treatment groups revealed no treatment-related evidence of systemic toxicity resulting from topical administration of the test material. The observations that were made on these organs were considered to be spontaneous and unassociated with dermal exposure to test material. The increase in liver weight was not associated with any histopathologic changes and therefore was not considered to be toxicologically significant.

Effect levels

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 systemic signs of toxicity were noted during the study.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Range-finder Results

In-life clinical signs of toxicity and abnormal dermal observations were not present in the rabbits which had a topical administration of 1000 mg test material/kg/ day for four consecutive days. In addition, there were no in-life observations noted during daily examinations which were indicative of a treatment-related effect.

Based on these results, 1000 mg/kg/ day was selected as the highest dose for the 21-day study with 500 and 100 mg/kg/ day selected to evaluate a potential dose-relationship.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the repeated dermal administration of test material resulted in local dermal irritancy in rabbits administered 100, 500 or 1000 mg/kg/ day. For systemic toxicity, the no observed-effect level (NOEL) was 1000 mg/kg/day, the accepted limit test level for male and female New Zealand White rabbits following dermal administration.

Executive summary:

The repeated dose toxicity of the test material, when administered via the dermal route, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 410, EPA OPP 82-2, MAFF and EEC Directive 67/548/EEC.

During the study groups of ten New Zealand White rabbits (5/sex/dose) received 15 applications of 0 (control), 100, 500 or 1000 mg test material/kg/day. Data were collected on the following: clinical appearance and behaviour; in-life and terminal body weights; dermal irritation at the test site; clinical chemistry and haematologic parameters; organ weights; and gross and histopathologic appearance of tissues.

There were no indications of systemic toxicity based on in-life clinical observations, body weights, liver, kidneys, and testes weights, clinical pathology parameters, gross pathology or histopathology of the liver and kidneys. During the in-life portion of the study, treatment-related dermal effects were observed. One male and two females in the 500 mg/kg/day group had slight to moderate scaling at the site of application.

Treatment-related histopathologic changes at the dermal test site were present in both sexes. Although there was an apparent increase in epidermal hyperplasia (very slight, or slight at most), and hyperkeratosis at the low dose and above, examination of control animals, and untreated skin on treated animals showed a certain back ground level of these effects. When it is considered that actual gross erythema and oedema do not appear to be dose related in either sex, and in the absence of any systemic toxicity at the limit dose (indicating no significant dermal penetration) the relevance of these histological finding as direct toxicologically significant findings becomes questionable. Where there is an apparent possible dose related increase in hyperkeratosis and epidermal hyperplasia in the absence of erythema or oedema, this may merely be a symptom from increasing degrees of dry skin, due to the absorptive properties of the increasing quantities of powder applied. Therefore as the histopathology is considered a result of the physical chemical (adsorptive properties) of the powder and not a toxicological effect.

Under the conditions of this study, 100 mg/kg bw/day may be taken as a conservative NOAEL for skin effects. The application site was 10 cm by 15 cm. The dose received/sq.cm by rabbits at 100 mg/kg = (3.3 kg x 100 mg/kg) / 150 sq.cm = 2.2 mg/sq.cm. For systemic toxicity, the no observed-effect level (NOEL) was 1000 mg/kg/day, the accepted limit test level for male and female New Zealand White rabbits following dermal administration.