Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
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
Reference
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
Reason / purpose for cross-reference:
reference to same study
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
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%
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
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.
Dose / conc.:
8 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle
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%.
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: 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.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
Key result
Dose descriptor:
NOAEL
Effect level:
8 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Sex:
male/female
Basis for effect level:
viability
Key result
Critical effects observed:
no
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

Data source

Referenceopen allclose all

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 Type:
other: Tables of study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD TG 421
Deviations:
yes
Remarks:
Dams with offspring killed on day 5 post-partum, instead of on day 13
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
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
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
Doses / concentrationsopen allclose all
Dose / conc.:
8 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle
Details on study design:
- 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.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS
Time schedule: Daily

BODY WEIGHT
- 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 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.
Ovaries and uterine content:
numbers of corpora lutea and implantation sites were examined
Fetal examinations:
number of live and dead pups, sex, external malformations on day 0 of lactation period
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.
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%.
- 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%.
Historical control data:
not reported

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- 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.
Mortality:
mortality observed, treatment-related
Description (incidence):
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 efficiency:
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
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- The following gross pathological findings were observed 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’): haemorrhage in the lumen of the uterus, incomplete retention and red colour in the lung, and dark red medulla and hardness on the kidney in both animals; hydrothorax in the thoracic cavity, attachment of red content in mucosa of the glandular stomach and recessed area, or red spots in the duodenum in either animal.
Neuropathological findings:
not examined
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

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The pup delivery index ((number of pups born / number of implantations) x 100) was dose-dependently decreased. This difference was not statistically significant.
There was no difference regarding pre-implantation losses (implantations index) between the treated groups and the control group.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
effects 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.
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
Gestation length was significantly increased at 50 and 300 mg/kg (in both groups by 0.6 days compared to control animals).
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Gestation length was significantly increased at 50 and 300 mg/kg (in both groups by 0.6 days compared to control animals).
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
8 mg/kg bw/day
Basis for effect level:
other: longer gestation length

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
effects 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 (see "any other information on results incl. tables").
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
effects 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 (see "any other information on results incl. tables").
External malformations:
no effects observed
Skeletal malformations:
not specified
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
reduction in number of live offspring
changes in postnatal survival

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Any other information on results incl. tables

Developmental findings in rats dosed with DPPD by gavage:

Dose (mg/kg bw/d)

0

8

50

300

Day 0 of lactation

Number of pups born

14.8 ± 2.1 (13)

14.8 ± 3.1 (12)

14.3 ± 1.5 (12)

13.7 ± 3.1 (11)

Delivery index

92.5 ± 7.5 (13)

90.7 ± 8.2 (12)

88.3 ± 8.7 (12)

86.7 ± 16.1 (11)

Number of live pups

14.7 ± 2.1 (13)

14.4 ± 2.7 (12)

13.8 ± 1.3 (12)

12.8 ± 4.1 (11)

Birth index

92.1 ± 7.9 (13)

88.4 ± 7.1 (12)

85.8 ± 10.1 (12)

81.2 ± 24.7 (11)

Live birth index

99.5 ± 1.7 (13)

97.7 ± 5.4 (12)

97.2± 5.3 (12)

92.0± 20.7 (11)

Day 4 of lactation

Number of live pups

14.5 ± 1.9 (13)

13.9 ± 2.6 (12)

13.8± 1.4 (12)

12.2± 5.0 (11)

Viability index

99.1 ± 2.2 (13)

97.0 ± 8.5 (12)

99.5± 1.8 (12)

87.5± 30.0 (11)

Parentheses indicate the number of dams.

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

Viability index = (number of live pups on day 4/number of live pups on day 0) x 100

Applicant's summary and conclusion