N,N'-diphenyl-p-phenylenediamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference 1

Reference Type:

publication

Title:

An antioxidant, N,N 0 -diphenyl-p-phenylenediamine (DPPD), affects labor and delivery in rats: A 28-day repeated dose test and reproduction/developmental toxicity test

Author:

Matsumoto M. et al.

Year:

2013

Bibliographic source:

Food and Chemical Toxicology 56 (2013) 290–296

Reference 2

Reference Type:

other: tables from study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: the notice on the test method concerning new chemical substances ( No. 1121002, Pharmaceutical and Food Safety Bureau, MHLW; No.2, Manufacturing Industries Bureau, METI; No. 031121002, Environmental Policy Bureau, MOE)

Version / remarks:

November 21, 2003

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: see version/remarks

Version / remarks:

the standard for the test facility conducting tests concerning new chemical substances, etc. (November 21, 2003, No. 1121003 Pharmaceutical and Food Safety Bureau, MHLW; No. 3 Manufacturing Industries Bureau, METI; No. 031121004 Environmental Policy Bureau, MOE)

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-diphenyl-p-phenylenediamine

EC Number:

200-806-4

EC Name:

N,N'-diphenyl-p-phenylenediamine

Cas Number:

74-31-7

Molecular formula:

C18H16N2

IUPAC Name:

N,N'-diphenyl-p-phenylenediamine

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot No. of test material: Seiko Chemical (Tokyo, Japan), 307605R
- Purity test date: 99.87%
- Name (as cited in article): DPPD

Test animals

Species:

rat

Strain:

other: [Crl:CD(SD)] SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Japan, Inc., (Kanagawa, Japan)
- Age at study initiation: Five weeks
- Weight at study initiation: male, 152-172 g; female, 130-147 g
- Housing: individually
- Diet: Basal diet, Labo MR Stock; NOSAN corporation (Tokyo, Japan), ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.9–23.0
- Humidity (%): 55–61
- Air changes (per hr): 10 and more
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on oral exposure:

Dosing volume: 5 mL/kg bw

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 other: mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 other: mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 other: mg/kg bw/day

No. of animals per sex per dose:

- control, 1000 mg/kg bw/day: 10
- 100, 300 mg/kg bw/day: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- After the dosing period, five rats per each sex at 0 and 1000 mg/kg bw/day were reared for 14 days without administration of DPPD as the recovery groups. The volume of each dose was adjusted to 5 mL/kg body weight based on the latest body weight.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Time schedule: Weekly

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Clinical signs in detailed observation in all animals were recorded one day before the administration period and once a week during the administration period.

BODY WEIGHT
- Time schedule for examinations: days 1, 7, 14, 21, and 28 of the administration period, days 7 and 14 of the recovery period, and on the day of necropsy.

FOOD CONSUMPTION
- Time schedule for examinations: Once a week during both administration and recovery periods.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: yes

HAEMATOLOGY
- Time schedule for collection of blood: day of euthanizing (1 day after final administration or 1 day after completion of the recovery period)
- Anaesthetic used for blood collection: Yes, for euthanizing by exsanguination
- Animals fasted: Yes
- Collected blood samples were examined for haematology by an automated haematology analyzer (XT-2000i, Sysmex Co., Kobe, Japan) and automatic coagulometer (KC-10A, Amelung, US). Serum biochemistry was tested by an automatic analyzer (JCA-BM8, JEOL, Tokyo, Japan) and automated electrolyte analyzer (NAKL-132, TOA electronics Ltd., Tokyo, Japan)

URINALYSIS
- Time schedule for collection of urine: Fresh urine was sampled from animals on day 22 of the administration period and on day 8 of the recovery period.
- Urine samples were tested for colour, pH, protein, glucose, ketone bodies, bilirubin, occult blood, and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: Day 27 of the administration period and day 13 of the recovery period.
- Battery of functions tested: Sensory reactions for a sight reaction, hearing reaction, sense of touch reaction, pain reaction, pupil reflex, and righting reflex; grip strength of fore and hind limbs and spontaneous motor activity

Sacrifice and pathology:

SACRIFICE
Euthanized by exsanguination, 1 day after final administration or 1 day after completion of the recovery period

GROSS PATHOLOGY
External surfaces of the rats were examined. Abdomen and thoracic cavities were opened, and gross internal examination was performed. The brain, thymus, heart, liver, spleen, kidney, adrenal gland, thyroid gland, pituitary gland, testis, epididymis, and ovary were isolated and weighed.

HISTOPATHOLOGY
Histopathological evaluations were performed on the organs mentioned in ‘gross pathology’ in addition to the eye ball, spinal cord, lung, trachea, stomach, intestines, prostate, seminal vesicle, vagina, uterus, urinary bladder, sciatic nerve, lymph nodes, and bone marrow (femur) in control and highest dose groups.

Other examinations:

- Bilirubin levels significantly increased without being related to toxicological effects in males. Because both bilirubin and the test compound contain – NH substitutes, the interference of DPPD with bilirubin measurements was anticipated.
- The interference of test compound with bilirubin measurements was tested as follows. Serum samples were taken from untreated male rats, and 0.2 mL of test compound at 0.001, 0.01, 0.1, and 1 mg/mL (1:1 aceton and dimethyl sulfoxide) was added to 0.5 mL serum of rats. In addition, rat liver S9 was added to test compound at 0.1 mg/mL to test the interference of test compound metabolites. Bilirubin levels were measured by the diazo method.

Statistics:

To assess the homogeneity of data, parametric data were analysed with Bartlett’s test or the F-test. When homogeneity was recognized, data were analysed using a one-way analysis of variance or the Student’s t-test. Non-homogeneous data were analysed with Kruskal–Wallis’s rank test or the Aspin–Welch t-test. Nonparametric data were analysed with Kruskal–Wallis’s rank test or Mann–Whitney’s U test. The Dunnett test or Dunnett type test was used to assess multiple comparisons. Fisher’s exact test was used to assess categorical data. Five per cent levels of probability were used as the criterion for significance.

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):

effects observed, non-treatment-related

Description (incidence and severity):

- Food consumption significantly decreased at 300 and 1000 mg/kg bw/day in males

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:

effects observed, treatment-related

Description (incidence and severity):

- Total bilirubin significantly increased in all treatment groups at the end of the treatment period in males, but it was not observed at the end of the recovery period. When the test compound was added to rat serum, bilirubin levels measured by the diazo method increased in a concentration-related manner with or without the rat S9 mix. Therefore, increased bilirubin levels in this study were considered to be due to interference by the test compound.
- In females, γ-GTP significantly decreased (0.63 IU/L) at 1000 mg/kg bw/day at the end of the administration period, but it was within the background data of the facility (0.31–2.06 IU/L) and was not considered to be toxicologically significant. This change was not observed at the end of the recovery period.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Protein levels significantly decreased in all treatment groups, but this was not dose dependent and was considered to be due to spontaneously occurring higher levels in control groups.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Relative and absolute weights of the thyroid gland in males and absolute weight of the kidney in females significantly increased at 100 mg/kg bw/day, but histopathological changes were not significantly different in these organs at the end of the administration period.
- No other effects were observed in organ weights in both sexes.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Slight hydrometra in the uterus was found in one female at 300 mg/kg bw/day, and dilatation of the lumen was histopathologically observed in the uterus of this female at the end of administration period; however, no gross or histopathological effects in the uterus were observed at 1000 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 other: mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Critical effects observed:

no