p-phenylenediamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Remarks:

The study was conducted according to guideline in effect at time of study conduct.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Purity: Not reported

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 40 days
- Weight at study initiation: the males weighed 145-184 g and the females weighed 127-174 g
- Housing: in groups of 5, by sex, in grid bottomed stainless steel cages, measuring 56 x 35 x 20 cm, suspended over cardboard lined excreta trays
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 25 - 60%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours light (0600 to 1800 hours) and 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF TEST SUBSTANCE FORMULATION:

- Solvent used: deionised, boiled water
- Preparation frequency: freshly each day for each dose level
- Preparation details & Adjusted for purity: The high dose level was prepared by dissolving a weighed quantity of test article in the appropriate volume of vehicle. Lower dose levels were prepared by dilution of the high dose formulation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method of Analysis: HPLC

Concentration verification (% of Target)): ±3%
Conducted on all dose levels: yes
Results: As a check on the accuracy of preparation, samples of each formulation prepared on one day during each of weeks 1 and 13 (including the control formulation) were analyzed for test article. In addition a sample was taken from all groups (including the control) once in each week. Samples were stored frozen (<18°C) for possible future analysis, until the Sponsor confirmed that all analytical results were satisfactory.
Weeks 1 & 13: The test substance was analyzed in water (purified and degassed) using a transferred method of analysis. The stability was assessed over a 2 day period; it was found that the test substance was stable up to 6 hours after preparation. Analysis of the study samples prepared in weeks 1 and 13 of the study indicated the solutions were prepared within ±3% of the nominal concentrations. This was considered to be acceptable for the purpose of the study.

Stability (% of Time Zero):
Conducted on dose levels: 0.5 and 4.0 mg/mL
Results in Weeks 1 & 13:
Nominal Concentration:
0.5 mg/mL = 102 (0 hours), 101.6 (4 hours), 101.5 (6 hours)
4.0 mg/mL = 95.3 (0 hours), 95.5 (4 hours), 95.4 (6 hours)
The stability of the test substance was assessed over a 2 day period at concentrations of 0.5 and 4.0 mg/mL. Formulated solutions were stored at ambient conditions in the dark. Samples were analyzed at 0, 1, and 2 days in the first trial and again at 0, 4, and 6 hours in the second trial. Stability was determined by the deviation from the day 0 result. The test article formulations of 4.0 mg/mL were found to be stable over 24 hours. Formulations of 0.5 mg/mL were not stable for 24 hours and hence a further stability trial was carried out over 6 hours, and formulations were found to be stable. As the low dose level chosen to be used on the study was at a lower concentration than that originally assessed during the stability trial a further stability trial was carried out over 4 and 6 hours, using the day 1 group 2 sample (0.2 mg/mL). 0.2 mg/mL was found to be stable for 6 hours.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
2, 4, 8, 16 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

15 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in light of the results from a range-finding study performed at Toxicol.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily for changes in condition or behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: Tthe first day of dosing, weekly thereafter, and at necropsy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes of all animals were examined before treatment started. In week 13 the eyes of all control and high dose animals were examined. The examinations were performed using a direct ophthalmoscope and then, if necessary, an indirect ophthalmoscope after previous instillation of a mydriatic agent (Minims - 2% w/v Homatropine hydrobromide).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 4 and 13. Blood samples were obtained by puncture of the lateral tail vein. Further blood samples were obtained during week 13 for assessment of clotting factors.
- Anaesthetic used for blood collection: not reported
- Animals fasted: Yes, but for further blood samples in week 13, the animals were not fasted.
- How many animals: All animals
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Weeks 4 and 13. Blood samples were obtained by puncture of the lateral tail vein. Further blood samples were obtained during week 13 for assessment of clotting factors.
- Anaesthetic used for blood collection: not reported
- Animals fasted: Yes, but for further blood samples in week 13, the animals were not fasted.
- How many animals: All animals
- Parameters checked in table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 4 and 12
- Metabolism cages used for collection of urine: not reported
- Animals fasted: Yes
- Parameters checked in table No. 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 3)

A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide asphyxiation. All animals were weighed and were then examined externally. A macroscopic examination was then performed by opening the cranial, thoracic and visceral cavities and by observing the appearance of the tissues in situ. The location, colour, shape and size of any abnormalities were recorded. The following tissues were dehydrated, wax embedded, cut at a nominal thickness of 5 microns, stained with haematoxylin and eosin and examined microscopically: all tissues from the control and high dose animals, all gross lesions from all animals, and lungs from all animals.

Statistics:

See Table 4

Results and discussion

Results of examinations

Clinical signs:

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):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

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:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no clinical signs noted that were considered to be related to administration of the test substance. Clinical signs that were noted included convulsions on 3 occasions in one female from group 2, and hunched posture, hypoactivity, pale extremities and irregular respiration on one day only in one female from group 4 (8 mg/kg). These were considered to be unrelated to test substance administration since no similar changes were observed in the highest dose group. Hair loss and scabbing were noted in both control and treated animals. There were no deaths during the treatment period.

BODY WEIGHT AND WEIGHT GAIN
Although a slight reduction (8%) in the bodyweight gain was noted in males from group 4 (8 mg/kg/day), all remaining groups (male and female) treated with the test substance (groups 2-5), had bodyweight gains similar to those of the controls. Therefore, this reduction was considered not to be treatment-related.

HAEMATOLOGY
There were no haematological changes noted that were considered to be related to administration of the test substance. During week 4 and 13, decreases in white blood cell counts in some, but not all of the treated groups, were noted. The changes were inconsistent and, as all the means were within the quoted reference ranges for this laboratory, were considered to be unrelated to treatment. A slight reduction in red blood cell count was present for both sexes from group 5 (16 mg/kg/day) during week 13 and females only during week 4, values were however within the normal range found in this laboratory. All other statistically significant changes were minor and within the normal reference ranges found in this laboratory.

CLINICAL CHEMISTRY
There were no changes in blood chemistry parameters that were considered to be related to test substance administration. Small increases in mean glucose levels were seen during the week 4 and week 13 examinations. Mean values were within the quoted reference range for both males and females. Although some of the individual glucose values were above our quoted reference ranges, as the differences were small, and not dose-related they are considered not to be toxicologically significant. Lactate dehydrogenase values (LDH) were variable. A statistically significant decrease in mean values was observed for males at the week 4 examination. At the week 13 examination some individual animals had values well above our quoted reference range. Lactate dehydrogenase is a variable parameter and these changes were considered to be too inconsistent to be associated with administration of the test substance.

ORGAN WEIGHTS
Absolute and bodyweight-related liver weights were statistically significantly increased for males from group 5 (16 mg/kg/day). A statistically significant increase was also observed for bodyweight-related liver weights for males from group 4 (8 mg/kg/day). Absolute and bodyweight-related kidney weights were increased for females from groups 4 and 5. As the increased body weight-related liver and kidney effects had no associated pathological changes (and are considered and adaptive response to dosing), they are not viewed as adverse. Absolute and bodyweight-related thyroid weights were statistically significantly increased for all treated male groups. The value for the control group was particularly low and in the absence of a dose response these findings were considered to be unrelated to test substance administration. Other statistically significant changes noted did not occur in the highest dose group and were therefore considered not to be of toxicological significance.


HISTOPATHOLOGY: NON-NEOPLASTIC
The small number of histological findings recorded was within the normal range of background alterations which may be seen in untreated rats of this age and strain.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at highest tested dose

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Male NOEL = 16 mg/kg/day
Female NOEL = 16 mg/kg/day

Executive summary:

One hundred and fifty rats of the Crl:CD(SD)BR strain (VAF plus), were divided into 5 groups each comprising 15 males and 15 females. Four groups received a solution of the test substance in deionised, boiled water, by gavage at dose levels of 2, 4, 8 or 16 mg/kg/day,whilst the fifth group received deionised, boiled water alone and acted as a control. All animals were dosed once daily, for 13 weeks, until the day before necropsy. During the study the animals were observed daily, bodyweights and food consumption were recorded weekly. Ophthalmoscopy was performed on all animals before the start of treatment and on control and high dose animals only during week 13. Blood and urine samples were obtained from all animals during weeks 4 and 12/13. At the end of the study all animals were killed and subject to necropsy, a range of organs was weighed and tissues examined microscopically.

 

All animals survived the treatment period. There were no treatment-related clinical signs, body weight changes, or effect on food consumption or ocular changes or abnormalities observed. There were no changes in haematological, blood chemical or urinalysis parameters that could be associated with administration of the test substance. The mean absolute and bodyweight-related liver weights were increased in males given 16 mg/kg/day compared to those of the controls. Bodyweight-related liver weights were also increased for males given 8 mg/kg/day. Mean absolute and bodyweight-related kidney weights were increased in females given 8 and 16 mg/kg/day compared to those of controls. There were no associated pathological changes noted for any of these organ weight changes. No treatment-related macroscopic or microscopic findings were noted.

 

Administration of the test substance by gavage at a dose level of 16 mg/kg/day was associated with increased mean absolute and bodyweight-related liver weights in males, and increased mean absolute and bodyweight-related kidney weights in females. There were, however no associated pathological changes. At 8 mg/kg/day, males had increased bodyweight-related liver weights and females had increased absolute and bodyweight related kidney weights. As the increased body weight-related liver and kidney effects had no associated pathological changes (and are considered and adaptive response to dosing), they are not viewed as adverse. Dose levels of up to 4 mg/kg/day did not produce any signs of response. In conclusion the no observed effect level (NOEL) for this study was 4 mg/kg/day and the NOAEL was 16 mg/kg/day.