N,N'-diphenyl-p-phenylenediamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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 et al.

Year:

2013

Bibliographic source:

Food and Chemical Toxicology 56 (2013) 290–296

Reference 2

Reference Type:

other: Tables of study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Dams with offspring killed on day 5 post-partum, instead of on day 13

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: Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Lot No. KWR0015
- Purity test date: 100%

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: 10 wks
- Weight at study initiation: Males: 370.2-446.9 g; Females: 220.4-265.2 g
- Housing: individually, except for mating and lactation periods
- Diet: CE-2; CLEA Japan, Inc. (Tokyo, Japan), ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5–23.5 (air-conditioned)
- Humidity (%): 47–67
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

- The volume of each dose was adjusted to 5 mL/kg body weight based on the latest body weight.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as [day 0] of pregnancy
- Further matings after two unsuccessful attempts: yes, mating period of two weeks

Duration of treatment / exposure:

- Males were dosed for a total of 42 days beginning 14 days before mating
- Females were dosed for a total of 42–46 days beginning 14 days before mating to day 4 of lactation throughout mating and gestation periods.

Frequency of treatment:

Daily

Details on study schedule:

- The first day of dosing was designated as day 1 of administration or day 1 of the premating period.
- The day on which parturition was completed by 11:00 was designated as day 0 of the lactation period.
- Once insemination was confirmed, females were checked for signs of parturition before 11:00 from day 21 of pregnancy.
- Females were allowed to deliver spontaneously and nurse their pups until day 5 of the lactation period.
- Litter size and numbers of live and dead pups were recorded, and live pups were sexed and individually weighed on days 0 and 4 of the lactation period. Pups were inspected for external malformations on day 0 of the lactation period.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

8 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

No. of animals per sex per dose:

13

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
Time schedule: Daily

BODY WEIGHT
- Time schedule for examinations (males): once a week during the administration period, and on the day of autopsy
- Time schedule for examinations (females): once a week during the pre-mating and mating periods, on days 0, 7, 14, and 21 of pregnancy, on days 0 and 4 of the lactation period and on a day of autopsy.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule males: Days 1–2, 7–8, 13–14, 29–30, 35–36, and 41–42 of the administration period.
- Time schedule females: Days 1–2, 7–8, and 13–14 of the pre-mating period, on days 0–1, 7–8, 14–15, and 20–21 of the pregnancy period, and on days 3–4 of the lactation period.

Oestrous cyclicity (parental animals):

Daily vaginal lavage samples of each female were evaluated for estrous cyclicity throughout the pre-mating period.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, live births, postnatal mortality, presence of external malformation, and weight gain.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: the day after the final administration (by exsanguination under anaesthesia).
- Maternal animals: day 5 of the lactation period (by exsanguination under anaesthesia).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations (thoracic, and abdominal viscera).

HISTOPATHOLOGY / ORGAN WEIGHTS
- Males: The testis, epididymis, prostate, and seminal vesicle were isolated, and the testis and epididymis were weighed and histopathologically examined.
- Females: The ovary, uterus, vagina, and mammary gland were isolated, and the ovary was weighed and histopathologically examined. The numbers of corpora lutea and implantation sites were counted.
- Organs were stored in 10% formalin with 0.1 M phosphate buffer. Organs that showed gross pathological changes were histopathologically examined.

Postmortem examinations (offspring):

SACRIFICE
The F1 offspring was sacrificed on day 5 of the lactation period (euthanized by exsanguination under anaesthesia)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

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. Statistical analysis of pups was carried out using the litter as the experimental unit in the reproductive/developmental study.

Reproductive indices:

CALCULATED INDICES
- Copulation index: (number of copulated pairs/number of mated pairs) x 100%.
- Fertility index: (number of fertile males/number of copulated pairs) x 100%.
- Implantation index: not specified.
- Delivery index: (number of pups born/number of implantations) x 100%.
- Birth index: (number of live pups on day 0/number of implantations) x 100%.
- Live birth index: (number of live pups on day 0/number of pups born) x 100%.

Offspring viability indices:

CALCULATED INDICES
- Sex ratio: (number of male live pups/number of live pups) x 100%.
- Viability index on day 4 of lactation: (number of live pups on day 4/number of live pups on day 0) x 100%.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Clinical toxicity was not observed in males
- At 300 mg/kg bw/day, two females showed piloerection, hypothermia, and pale skin on day 23 of pregnancy. One of these two females died and the other was sacrificed due to dystocia on day 23 of pregnancy. Another female showing piloerection and pale skin delivered only three live pups. Nesting and nursing were not observed in this female, and this female was sacrificed on day 1 of lactation due to total litter loss.
- At 300 mg/kg bw/day, one female showed piloerection on day 23 of gestation, and another female showed pale skin on day 22 of gestation. However, no abnormalities were found in their delivery.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

- Males: Mortality was not observed
- Females: on day 23 of pregnancy one female showing clinical signs died, another was sacrificed due to dystocia on day 23 of pregnancy. On day 1 of lactation one female was sacrificed due to total litter loss.

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:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- The following observations were made in two females who died or were sacrificed on day 23 of pregnancy (300 mg/kg bw/day, for details see ‘description’ under ‘clinical signs’): slight haemorrhage in the endometrium, and very slight edema, very slight foam cell accumulation in alveolus, and very slight capillary fibrinous thromboses in the lung were observed in the two females.
- The histopathological examination revealed no toxicological effects in other males and females.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- One female at 8 mg/kg bw/day did not deliver pups by day 25 of gestation. An autopsy on day 26 of gestation revealed no implantations in this female.
- No changes attributable to the chemical were noted in the number of mated pairs, number of copulated pairs, copulation index, number of fertile males, fertility index, length of estrus cycle, pairing days until copulation, number of corpora lutea, number of implantations, implantation index, and number of pregnant females.
- Gestation lengths were significantly longer than the control group at 50 and 300 mg/kg bw/day.
- Although no statistical significance was observed, the number of pups born, delivery index, number of live pups, birth index, and live birth index on day 0 of lactation dose dependently decreased.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

2

Key result

Dose descriptor:

NOAEL

Effect level:

8 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Although no statistical significance was observed, the number of live pups and viability index were decreased on day 0 and day 4 of lactation in treatment groups, especially at 300 mg/kg bw/day.

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:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Sex:

male/female

Basis for effect level:

viability

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified