Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 July 1981 - 22 February 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Non-GLP study; however study and results are well documented with full statistical analysis included.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objectives of the study were to evaluate the influence of the dietary administration of 4,4'-oxydianiline on the reproduction/lactation function of one generation of CD® rats during the production of two litters of pups and to investigate previous observations of 4,4'-oxydianiline-related testicular lesions produced in male Fischer 344 rats in a 90-day feeding study.

Weanling rats were administered 4,4’-oxydianiline in their feed for 90 days.

> Male Fischer 344 rats:
During the 90-day feeding phase, body weights, clinical observations, and individual food consumption were recorded.
After approximately 90 days of continuous feeding, all Fischer 344 rats were sacrificed and the reproductive tracts were examined for gross abnormalities. Testes with epididymides were weighed and relative testes weights were calculated. Tissues processed for histopathological examination consisted of testis, epididymis, prostrate (ventral and dorsal), seminal vesicle, coagulating gland, ampullary gland, and urinary bladder.

> Male and female CD rats
Following the 90-day feeding study, all surviving CD rats (F0) were used in a 1-generation, 2-litter reproduction study. The rats were mated in the following manner:
* 10 males from the 10, 100, and 400 ppm groups were mated to untreated females;
* 10 females from the 10, 100, and 400 ppm groups were mated to untreated males;
* 10 untreated males were mated to 10 untreated females;
* 10 males from the test groups were mated to females from the corresponding test groups.
On day 4 postpartum, the litters (F1A) were culled randomly to 10. Remaining pups were sacrificed and did not receive pathological evaluation. Weanlings (21 days after birth) were weighed, sexed, and sacrificed, and did not receive pathological evaluation. Pups that died prior to weaning or F0 females that died during the reproduction phase of the study did not receive pathological evaluation.
Approximately 1 week after weaning the last F1A litter, the F0 females were mated again in the same manner as described above, but to different F0 males (from the same group as previous mating) to produce the F1B litters.
Following the last mating in the reproduction phase of the study, the male F0 CD rats were sacrificed, and testes with epididymides were weighed and the reproductive tract was examined for gross abnormalities, as described previously for the Fischer 344 rats.
F1B pups were treated in the same manner as the F1A pups. After weaning of the F1B pups, all F0 females were sacrificed and did not receive pathological evaluation.
During the reproduction phase of the study, the following were recorded: all matings, number of females bearing litters, number of pups born and born alive, individual litter weights 24 hours and 4 days postpartum, number of pups before and after litter reduction, number of pups per litter 12 days postpartum, number and individual body weights of male and female pups at weaning, and body weights of F0 female rats at the time of weaning of their pups. The following reproduction and lactation parameters were determined: fertility, gestation, viability, and lactation indices, mean number of pups per litter, percent of pups born alive, litter survival, mean pup weights per litter, and mean male and female weanling body weights per litter.
During the reproduction phase of the study all F0 male and female rats and all litters were examined at least once daily for abnormal behavior or appearance and mortality.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
light pink to white solid material
Details on test material:
- Name of test material (as cited in study report): 4,4'-Oxydianiline
- Analytical purity: stated to be 98.7% pure
- Lot/batch No.: Assigned Haskell Laboratory identification number 14108
- Physical state: light pink to white solid material
- Molecular formula (if other than submission substance): Not specified.
- Molecular weight (if other than submission substance): Not specified.
- Smiles notation (if other than submission substance): Not specified.
- InChl (if other than submission substance): Not specified.
- Structural formula attached as image file (if other than submission substance): Not specified.
- Substance type: Organic
- Impurities (identity and concentrations): Not specified.
- Composition of test material, percentage of components: Not specified.
- Isomers composition: Not applicable.
- Purity test date: Not specified.
- Expiration date of the lot/batch: Not specified.
- Radiochemical purity (if radiolabelling): Not applicable.
- Specific activity (if radiolabelling): Not applicable.
- Locations of the label (if radiolabelling): Not applicable.
- Expiration date of radiochemical substance (if radiolabelling): Not applicable.
- Storage condition of test material: Not specified.
Specific details on test material used for the study:
4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4

Test animals

Species:
rat
Strain:
other: Fischer 344 and CD strains both used.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Fischer 344 (Males): Sprague-Dawley, Madison, WI
CD (Males / Females): Charles River Breeding Laboratories, Wilmington, MA

- Age at study initiation:
Fischer 344 (Males): 6 weeks
CD (Males / Females): 6 weeks

- Weight at study initiation:
Fischer 344 (Males): 129.3 - 130.3 g

CD
Males: 171.4 - 173.1 g
Females : 142.9 - 144.8 g

- Fasting period before study: None

- Housing: Stainless steel wire-mesh cages suspended above DACB® Deotized Animal Cage Boards, four to five animals per cage, of the same sex within a strain. On the sixth day of the pretest period, rats were housed in pairs of the same sex and strain and identified temporarily by colored tail markings and cage housing numbers. Fischer 344 rats were ear-punched.

- Use of restrainers for preventing ingestion (if dermal): Not applicable.

- Diet (e.g. ad libitum): ground certified Purina® Rodent Chow #5002 diet ad libitum until Day 29, then 1600 - 0800 timeframe per day.

- Water (e.g. ad libitum): tap water ad libitum

- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS: Not specified; standard laboratory conditions assumed.
- Temperature (°C): Not specified.
- Humidity (%): Not specified.
- Air changes (per hr): Not specified.
- Photoperiod (hrs dark / hrs light): Not specified.

IN-LIFE DATES: From: 23 July 1981 To: 22 February 1982

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): For the first 27 days of the study, control and test diets were prepared fresh weekly and stored under refrigeration until used.
However, due to concerns regarding stability of the test material in the diet under these storage conditions, from test day 28 to the end of the study, control and test diets prepared weekly were stored frozen until used.

- Mixing appropriate amounts with (Type of food): All test diets contained 1% (w/w) final concentration of Mazola® Corn Oil to ensure a homogeneous mixture of test material in ground certified Purina* Rodent Chow diet. Control diets also contained 1% (w/w) final concentration Mazola® Corn Oil

- Storage temperature of food: Specified as refrigerated until Day 27 of the study; from Day 28 onwards, frozen.
Details on mating procedure:
- M/F ratio per cage: 1: 1

- Length of cohabitation: 15 days

- Proof of pregnancy: Copulation plug referred to as day 1 of pregnancy

- Further matings after two unsuccessful attempts: Not specified.

- After successful mating each pregnant female was caged: Housed individually in polypropylene cages wit;h Ad-sorb-dri® cage bedding

- Additional Information:
Following the 90-day feeding study, all surviving CD® rats were used in a one-generation, two-litter reproduction study.
The following mating schedule was conducted:
* 10 males from treatment dose groups 10 ppm, 100 ppm, and 400 ppm were mated to untreated females;
* 10 females from dose groups 10 ppm, 100 ppm, and 400 ppm were mated to untreated males;
* 10 untreated males were mated to 10 untreated females;
* 10 males from the test groups were mated to females from the corresponding test groups.
During the first 15-day mating phase, each female (F0) was housed with one male. All males and females were fed ground certified Purina® Rodent Chow #5002 diet during the mating phase.

After completion of the mating phase, female rats were separated from 'male rats and housed individually in polypropylene cages with Ad-sorb-dri® cage bedding. Following separation, the male and female rats received their respective treatment group's diet.

Six days after separation from the males, the female rats were examined twice daily for birth of young (F1A).
Litter weights were obtained approximately 24 hours and four days after birth.
Litters that contained more than ten pups were reduced to this number four days after birth by random selection.
Remaining pups were sacrificed and discarded without pathological evaluation.
Twenty-one days after birth, surviving pups were weighed, sexed, sacrificed, and discarded without pathological evaluation.
Pups that died prior to weaning (21 days postpartum) or F0 females that died during the reproduction phase of the study were discarded without pathological evaluation.

Approximately one week after weaning the last F1A litter, the F0 females were mated again in the same manner as described above, but to different F0 males (from the same group as previous mating) to produce the F1B litters.
During the 15-day mating period, each female was checked daily for the presence of copulation plugs.
Following mating for the F1B litters, the F0 males were sacrificed and subjected to pathological evaluation.
F1B pups were treated in the same manner as the F1A pups. After weaning of the F1B pups, all F0 females were sacrificed and discarded without pathological evaluation.

During the reproduction phase of the study, records of the following data were kept:
all matings, number of females bearing litters, number of pups born and born alive in each litter, individual litter weights 24 hours and four days postpartum, number of pups before and after litter reduction (four days postpartum), number of pups per litter 12 days postpartum, number and individual body weights of male and female pups at weaning (21 days postpartum), and body weights of EQ female rats at the time of weaning of their pups.
From the appropriate data, the following reproduction and lactation parameters were determined: fertility, gestation, viability, and lactation indices, mean number of pups per litter, percent of pups born alive> litter survival, mean pup body weights per litter 24 hours and four days postpartum, and mean male and female weanling body weights per litter,

During the reproduction phase of the study, all F0 male and female rats and all litters were examined at least once daily for abnormal behavior or appearance and mortalities, pups were allowed access to their respective group's diet, when available, and tap water ad libitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All samples were analyses within the laboratory using HPLC techniques.
The concentration of 4,4’-oxydianiline in diet samples collected and frozen immediately after mixing ranged from 92-95% of the nominal dose levels. Lower concentrations, ranging from 76-92% 4,4’-oxydianiline, were detected in samples stored under refrigeration for 3 or 7 days. Concentrations detected in samples stored at room temperature for 16 hours, 24 hours, or 7 days were 80-91%, 78-87%, and 57-67%, respectively. The data suggested instability or binding of 4,4’-oxydianiline to rodent chow when diets were refrigerated or stored at room temperature. To minimize this effect, rats were fed daily from frozen diets and allowed limited access to the diets from test day 30 through the end of the study. Concentrations of 4,4’-oxydianiline measured in diet samples which simulated actual in-use conditions (i.e., frozen diets stored at room temperature for 16 hours) ranged from 80-91% of the nominal diet concentrations. Since diet samples were not analyzed for impurities, the available data are insufficient to determine if the differences between nominal and analytical values of 4,4’-oxydianiline were due to instability or binding of the test material to rodent chow.
Duration of treatment / exposure:
> male CD: 6 months
> female CD: 7 months
> male fisher 344: 3 months

> During the mating phases (15-days) rats (male and female CD) where fed ground certified Purina® Rodent Chow #5002 diet without the relevant dose. Following separation, the male and female rats received their respective treatment group's diet
Frequency of treatment:
For the first 29 days of the study, all rats received their respective group's diet ad libitum.
However, due to the concerns regarding test material stability, from test day 30 to the end of the study, the rats were given access to their respective group's diet from approximately 16h hours, daily.
Details on study schedule:
Following the 90-day feeding study, all surviving CD rats (F0) were used in a 1-generation, 2-litter reproduction study.
The rats were mated in the following manner:
* 10 males from the 10, 100, and 400 ppm groups were mated to untreated females;
* 10 females from the 10, 100, and 400 ppm groups were mated to untreated males;
* 10 untreated males were mated to 10 untreated females;
* 10 males from the test groups were mated to females from the corresponding test groups.

During the 1st 15-day mating phase, each female was housed with 1 male. After completion of the mating phase, female rats were separated from male rats and individually housed. Six days after separation from the males, the females were examined twice daily for birth of young (F1A). On day 4 postpartum, the litters were culled randomly to 10. Remaining pups were sacrificed and did not receive pathological evaluation. Weanlings (21 days after birth) were weighed, sexed, and sacrificed, and did not receive pathological evaluation. Pups that died prior to weaning or F0 females that died during the reproduction phase of the study did not receive pathological evaluation.

Approximately 1 week after weaning the last F1A litter, the F0 females were mated again in the same manner as described above, but to different F0 males (from the same group as previous mating) to produce the F1B litters. During the 15-day mating period, each female was checked daily for the presence of copulation plugs.
Following the last mating in the reproduction phase of the study, the male F0 CD rats were sacrificed, and testes with epididymides were weighed and the reproductive tract was examined for gross abnormalities, as described previously for the Fischer 344 rats. F1B pups were treated in the same manner as the F1A pups. After weaning of the F1B pups, all F0 females were sacrificed and did not receive pathological evaluation.

During the reproduction phase of the study, the following were recorded: all matings, number of females bearing litters, number of pups born and born alive, individual litter weights 24 hours and 4 days postpartum, number of pups before and after litter reduction, number of pups per litter 12 days postpartum, number and individual body weights of male and female pups at weaning, and body weights of F0 female rats at the time of weaning of their pups. The following reproduction and lactation parameters were determined: fertility, gestation, viability, and lactation indices, mean number of pups per litter, percent of pups born alive, litter survival, mean pup weights per litter, and mean male and female weanling body weights per litter.
During the reproduction phase of the study all F0 male and female rats and all litters were examined at least once daily for abnormal behavior or appearance and mortality.
Doses / concentrationsopen allclose all
Dose / conc.:
10 ppm (nominal)
Remarks:
in diet
Dose / conc.:
100 ppm (nominal)
Remarks:
in diet
Dose / conc.:
400 ppm (nominal)
Remarks:
in diet
No. of animals per sex per dose:
Fischer 344 Rat CD® Rat
Treatment Group Male Male Female
Control 10 40 40
Low Dose (10 ppm) 10 20 20
Intermediate Dose (100 ppm) 10 20 20
High Dose(400 ppm) 10 20 20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
Doses were selected on the basis of previous studies performed to assess long term toxicity of the test substance in rats.

4,4'-Oxydianiline has slight oral acute toxicity; its approximate lethal dose (ALD) in young, adult CD® male rats was 1,500 mg/kg of body weight.
Clinical signs of toxicity observed in rats administered lethal doses of 4,4'-oxydianiline consisted of inactivity, lacrimation, body weight loss, prostration, pale and glassy eyes, and shallow respiration. Sublethal doses (200-1,000 mg/kg) of 4,4'-oxydianiline produced hair loss, body weight loss, and evidence of extraraedullary hematopoiesis. Oral administration of a single dose of 90 mg 4,4'-oxydianiline/kg of body weight caused no apparent toxic effects.

A NCI-sponsored ninety-day feeding study was conducted at Mason Research Institute in which male and female Fischer 344 rats were fed diets that contained 0, 3, 10, 30, 100, or 300 ppm 4,4'-oxydianiline. No compound-related adverse effects were observed in the treated rats. The study was repeated at higher dose levels which consisted of 0, 300, 600, 1,000 or 2,000 ppm 4,4'-oxydianiline. A dose-related decrease in body weight gain in male and female rats fed diets that contained 600, 1,000 or 2,000 ppm 4,4'-oxydianiline was observed. Clinical signs of toxicity noted in rats fed 1,000 and 2,000 ppm 0DA consisted of alopecia, dyspnea, lethargy, and cyanosis. Administration of diets that contained 1,000 and 2,000 ppm 4,4'-oxydianiline was associated with 40 and 70% mortality, respectively, in female rats.
Gross pathological examination of reproductive tract tissues from male Fischer 344 rats in the 2,000 ppm 4,4'-oxydianiline group indicated atrophy of the gonads and seminal vesicles. Histopathological examination showed marked degeneration of the seminiferous tubules in the 600, 1,000 and 2,000 ppm groups; aspermia was common and the tubular lumina contained exfoliated epithelial cells and multinucleated giant cells. The germinal epithelium was decreased, disorganized, or missing, and one to two rows of Sertoli cells lined the tubules. Slight tubular changes were evident in nine of ten rats in the 300 ppm group. In addition, atrophy of the prostate gland and seminal vesicles in seven of ten rats in the 1,000 ppm group and ten of ten rats in the 2,000 ppm group was observed.

A long-term study was conducted in which male and female rats were fed diets which contained 0, 200, or 400 ppm 4,4'-oxydianiline for 23 months. The final mean body weights of male and female rats fed diets that contained 400 ppm 4,4'-oxydianiline and of male rats fed diets that contained 200 ppm 4,4'-oxydianiline were significantly decreased when compared to their respective control group. An apparent dose-related decrease in the time to onset of eye-related clinical signs was also noted. Male rats fed diets that contained 200 and 400 ppm 4,4'-oxydianiline exhibited a significantly-higher incidence of testicular tumours (age-adjusted). Male rats fed diets that contained 200 ppm 4,4'-oxydianiline had a higher incidence of testicular arteritis and focal interstitial ceil hyperplasia than the controls; however, the incidence of the lesions in the male 400 ppm group was comparable to that of control males.

- Rationale for animal assignment (if not random):
Test subjects were selected on the basis of body weight gain and freedom from any clinical signs of disease or injury. 40 Fischer 344 rats and 100 male and 100 female CD® rats were selected and divided by computerized-stratified randomization into the respective test groups.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily / One daily during reproductive phase.
- Cage side observations were not recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Not specified with regards to timeframe. Observations were documented, and were generally unremarkable.

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable.
Oestrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Not determined.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1A / F1B ] offspring:
Mean number of pups per litter, percent of pups born alive, litter survival, mean pup body weights per litter 24 hours and four days postpartum, and mean male and female weanling body weights per litter

GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: After approximately 90 days of continuous feeding, all Fischer 344 rats were sacrificed and the reproductive tracts were examined for gross abnormalities.
Following the last mating in the reproduction phase of the study, the male F0 CD rats were sacrificed.
- Maternal animals: After weaning of the F1B pups, all F0 females were sacrificed and discarded without pathological evaluation

GROSS NECROPSY
- Males (Fischer 344 rats and CD rats): Following sacrifice, testes with epididymides were weighed and, with final body weight data, the relative testes weights were calculated (expressed as percent body weight). In addition, gross examinations on reproductive tracts (testes, seminal vesicles and prostate gland) were conducted.
- Females: discarded without pathological evaluation

HISTOPATHOLOGY / ORGAN WEIGHTS
- Males (Fischer 344 rats and CD rats): Tissues processed for histopathological examination consisted of testes, epididymides, prostates (ventral and dorsal), seminal vesicles, coagulating glands, ampullary - glands, and urinary bladder.
- Females: Discarded without pathological evaluation
Postmortem examinations (offspring):
SACRIFICE
- The F1A and F1B offspring were sacrificed at 21 days of age.
- These animals were discarded and not subjected to postmortem examinations.

GROSS NECROPSY
- Gross necropsy of the pups was not conducted.

HISTOPATHOLOGY / ORGAN WEIGTHS
Histopathological examination of the pups was not conducted.
Statistics:
Body and organ weight data were subjected to an analysis of variance.
The least significant difference (LSD) and/or Dunnett statistics were calculated whenever the ratio of variances (F-ratios) indicated that significant differences existed among groups.
Incidence of pathological findings and clinical observations were subjected to Fisher's Exact Test when appropriate.
Reproduction and lactation indices, mean pup body weights per litter 24 hours and four days postpartum, and mean male and female weanling body weights per litter among control and test groups were compared by the Mann-Whitney U Test.
Significance was judged at p<0.05 level of probability.
Reproductive indices:
The following calculations were used for generation of reproduction and lactation indices:

Fertility Index (%)a = ( total number of litters delivered / total number of females mated) per group x 100

Fertility Index (%)ab = ( total number of litters delivered to females observed with copulation plugs / total number of females observed with copulation plugs) per group x 100

Gestation Index (%) = (total number of females bearing litters with at least one live pup / total number of females bearing litters) per group x 100

Percent of Pups Born Alive Per Litter = (number of pups born alive / number of pups born) x 100

Viability Index Per Litter (%) = (number of pups alive four days postpartum / number of pups born alive) x 100

Lactation Index Per Litter (%) = [number of pups alive at weaning (21 days postpartum) / number of pups alive after litter reduction (4 days postpartum)] x 100

a Excluding female rats that died during the mating phase or before the last litter of that generation's test group was delivered.

b This calculation was used to determine fertility index for second mating
Offspring viability indices:
The following calculations were used for generation of reproduction and lactation indices:

Litter Survival (%) = (total number of litters at weaning / total number of litters delivered) per group x 100

Mean Number of Pups Per Litter = number of alive pups at birth in each litter / number of litters delivered

Mean Pup Bodyweight per litter at 24 hours or 4 days Postpartum = (Litter weight / number of pups in all litters in group) / number of litters in a group

Mean Male or Female Weanling Body Weight = (Total weight of male or female weanlings in litter / number of male or female weanlings in litter for all litters in a group) / number of litters in a group.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Three females rats in the 400 ppm group exhibited hypersensitivity to touch approximately two weeks after birth of the F1A litters. This clinical obsrevation however, was not seen at any other time for the remainder of the reproduction phase.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One F0 female rat in the 100 ppm group died during the delivery of the F1A litter. Another F0 female rat in the 100 ppm grop died the last day of the mating period for the F1B litter.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During test weeks one through three, the mean body weights of male CD® rats in the 400 ppm group were significantly lower than those of the male control group. However, when evaluated over the entire 13-week feeding interval, the mean body weight gain of the male CD rats in the 400 ppm group was comparable to that of the male controls.

Mean body weights of female CD rats in the 400 ppm group were significantly lower than those of the female controls throughout the study. Overall mean body weight gain exhibited by the female 400 ppm group was approximately 26% below that of the female control group. Although mean body weights of the female 100 ppm group were comparable to those of the female controls during the study, overall mean body weight gain was approximately 7% below that of the female control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Among female CD rats, the 400 ppm group consumed slightly less diet than did female control rats during the 90-day feeding period.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Female CD rats in the 400 ppm treatment group exhibited decreased food efficiency values when compared to the female control.
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In one CD rat in the 400 ppm group, histological examinations of the reproductive tract revealed a diffuse, severe unilateral , atrophy of the seminiferous tubules with only sertoli cells remaining in the wall of the tubules and a paucity of spermatogenesis. However no evidence of necrosis or degeneration was observed. Since the atrophy was unilateral, and similar though less severe changes were observed in control as well as treated rats, the alteration was not considered to be related to the substance.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
> Mating of untreated males to treated females:
Statistically-significant decreases in the mean number of pups per litter were observed in both the F1A and F1B litters after the mating of female rats in the 400 ppm group with untreated male rats.
Among F1B litters, a statistically-significant decrease in the mean number of pups born per litter was observed when female rats in the 100 ppm group were mated to untreated male rats.

> Mating of treated males to treated females:
The mean number of pups per litter was less in both F1A and F1B when compared to those produced by control group rats. A statistically-significant decrease in the mean number of pups per F1B litter was also observed after mating males and females from the 10 ppm groups, but not after the mating of male and female rats from the 100 ppm groups. However in absence of consistency, reproducibility and dose-rekatedness, the observations wre not conisdered substance-related.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
90-DAY FEEDING STAGE
Clinical observations made in male and female CD and male Fischer 344 rats during the 90-day feeding phase were generally unremarkable and no meaningful differences in the incidence of clinical observations were apparent among 4,4'-oxydianiline-treated and control rats during the 90-day feeding phase.
No mortalities occurred during the 90-day feeding phase.

REPRODUCTION STAGE:
The clinical observations seen were unremarkable and no meaningful differences in the incidences of clinical observations were apparent among control and treated rats during the reproduction phase of the study. Three female rats in the 400 ppm group exhibited hypersensitivity to touch approximately two weeks after birth of the F1A litters; this clinical observation, however, was not seen at any other time for the remainder of the reproduction phase.
Between days 4 and 12 after birth of the first litter, one female in the 400 ppm group that was mated to an untreated male was observed cannibalizing pups in the litter.

One F0 female rat (Animal #307084) in the 100 ppm group died during the delivery of the F1A litter.
Another F0 female rat (Animal #307091) in the 100 ppm group died the last day of the mating period for the F1B litter.No mortalities in the F0 male groups occurred during the reproduction phase of the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During test weeks one through three, the mean body weights of male CD® rats in the 400 ppm group were significantly lower than those of the male control group. However, when evaluated over the entire 13-week feeding interval, the mean body weight gain of the male CD rats in the 400 ppm group was comparable to that of the male controls. Mean body weights and weight gains of the male CD rats in the 10 and 100 ppm groups were, in general, comparable to those of the male control group throughout the study.

Mean body weights of female CD rats in the 400 ppm group were significantly lower than those of the female controls throughout the study. A transient weight loss seen between test days 63 and 70 in the female 400 ppm group was considered the result of an improper water line connection and consequent dehydration. Overall mean body weight gain exhibited by the female 400 ppm group was approximately 26% below that of the female control group. Although mean body weights of the female 100 ppm group were comparable to those of the female controls during the study, overall mean body weight gain was approximately 7% below that of the female control group, Mean body weights and weight gains exhibited by the female 10 ppm group during the study were comparable to those of the female control group.

Mean body weights and weight gains of all male Fischer 344-treated rats were comparable to those of the male Fischer 344 control rats throughout the study.

No meaningful differences in diet consumption were observed among male CD and Fischer 344 rats in the respective control and 4,4'-oxydlaniline-treated groups during the study.
Among female CD® rats, the 400 ppm group consumed slightly less diet than did female control rats during the 90-day feeding period. Diet consumed by the female 10 and 100 ppm groups was, in general, comparable to that of the female controls.

Food efficiency values for male CD® and Fischer 344 rats were comparable to those of the respective control group rats. Female CD® rats in the 400 ppm treatment group exhibited decreased food efficiency values when compared to the female control rats. Food efficiency values for the female 10 and 100 ppm groups were comparable to those of the female controls.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
There were slight differences between the strains and sexes in the daily intake of 4,4'-oxydianiline over time; these were related to differences in the rate of body weight gain and the amount of diet consumed during the study.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): Not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): Not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
> Mating of treated male to untreated females:
No meaningful differences in fertility, gestation, viability, or lactation indices for F1A and F1B litters produced by mating treated males to untreated females were observed between control and test groups. Similarly, no meaningful differences in the percent of live pups born per litter, mean number of pups per litter, litter survival, mean pup body weights per litter 24 hours and four days postpartum, or mean male and female weanling body weights In both F1A and F1B litters produced by the mating of treated males to untreated females were observed between control and test groups.

> Mating of untreated males to treated females:
In contrast to observations following the mating of treated male to untreated female rats, statistically-significant decreases in the mean number of pups per litter were observed in both the F1A and F1B litters after the mating of female rats in the 400 ppm group with untreated male rats. Among F1B litters, a statistically-significant decrease in the mean number of pups born per litter was observed when female rats in the 100 ppm group were mated to untreated male rats. Mean pup body weights per litter 24 hours and four days postpartum following both matings of the female 10, 100, and 400 ppm groups with untreated males were comparable to those of litters produced by the mating of control rats. In the F1A litters, a statistically-significant decrease in both the mean male and female weanling body weights per litter was observed when 400 ppm group females were mated with untreated males ; in the F1B litters, a decrease in mean weanling body weight per litter was seen only in the female pups.

> Mating of treated males to treated females:
The mean number of pups per litter was less in both the F1A and F1B litters when litters produced by males and females from the 400 ppm groups were compared to those produced by control group rats. A statistically-significant decrease in the mean number of pups per F1B litter was also observed after mating males and females from the 10 ppm groups, but not after the mating of male and female rats from the 100 ppm groups. Mean pup body weights per litter 24 hours and four days postpartum and mean male weanling body weights per litter were comparable between treated -and control matings. Mean female weanling body weight per litter was significantly decreased in F1A litters produced by male and female rats in the 400 ppm groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The mean absolute testes weight of Fischer 344 rats fed for 90 days with diets that contained 400 ppm 4,4'-oxydianiline was significantly lower than that of the Fischer 344 controls. The mean absolute and relative testes weights of Fischer 344 rats in the 10 and 100 ppm groups and the mean relative testes weight of Fischer 344 rats in the 400 ppm group were comparable to those of the Fischer 344 controls. In all treated CD rats, the mean absolute and relative testes weights were comparable to those of the CD control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
As above for organ weights. Females were discarded without pathological examination.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histological examination of the reproductive tracts of Fischer 344 rats fed for three months or CD rats fed for six months with diets that contained 10, 100, or 400 ppm 4,4'-oxydianiline revealed no lesions that could be related to the dietary administration of 4,4'-oxydianiline. In one CD rat in the 400 ppm group, histological examination of the reproductive tract revealed a diffuse, severe unilateral, atrophy of the seminiferous tubules with only Sertoli cells remaining in the wall of the tubules and a paucity of spermatogenesis; however, no evidence of necrosis or degeneration was observed.Since the atrophy was unilateral, and similar though less severe changes were observed in control as well as treated rats, the alteration was not considered to be related to the dietary administration of 4,4'-oxydianiline.

OTHER FINDINGS (PARENTAL ANIMALS)
None documented.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 ppm
Based on:
act. ingr.
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Generation: maternal and offspring F1a F1b
Key result
Dose descriptor:
LOAEL
Effect level:
400 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Generation: maternal and offspring F1a F1b

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
> Mating of treated female rats with untreated male rats:
In the F1A litters, a statistically significant decrease in both the mean male and female weanling body weights per litter was observed when 400 ppm group females were mated with untreated males. In the F1B litters, this decrease in mean weanling body weight per litter was seen only in the female pups.

> Mating of treated females rats with treated male rats:
Mean female weanling body weight per litter was significantly decreased in F1A litters produced by treated male and female rats in the 400 ppm groups.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One pup in the F1A litter from the mating of male and female rats in the 400 ppm group was born without a leg and tail.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Decreased litter sizes were observed in one of four litters produced by matings of female rats in the 10 and 100 ppm treatment groups. However, based on the absence of consistency, reproducibility, and dose-relatedness, the observations were not considered related to the dietary administration of 4,4'-oxydianiline.

CLINICAL SIGNS (OFFSPRING): Not determined.

BODY WEIGHT (OFFSPRING)
Female weanlings in litters produced from matings of female rats in the 400 ppm treatment group exhibited decreased mean body weights. This effect on pup growth may have been a direct compound effect resulting from ingestion by the pups of diet or milk that contained 4,4'-oxydianiline or an indirect compound effect on lactation which could have consisted of decreased quantity or altered nutritional value of milk. Although an indirect compound effect on lactation cannot be excluded, the decrease in pup body weights was seen only at weaning and only in the female offspring. The decrease in body weight gain observed in the maternal rats in the 400 ppm treatment group during a growth phase suggests a direct compound effect on the growth of female pups. Since the effect was only observed at weaning, the decreased weight was probably a result of ingestion of 4,4'-oxydianiline in the diet instead of milk.

SEXUAL MATURATION (OFFSPRING): Not determined

ORGAN WEIGHTS (OFFSPRING) : Not determined

GROSS PATHOLOGY (OFFSPRING) : Not determined

HISTOPATHOLOGY (OFFSPRING) : Not determined

OTHER FINDINGS (OFFSPRING): One pup in the F1A litter from the mating of male and female rats in the 400 ppm group was born without a leg and tail.

Effect levels (F1)

Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 ppm
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects

Any other information on results incl. tables

The following tabulated data are included below under "attachments" for reference.

Table Ref

ParameterDocumented

I

Percent of Nominal Concentration of 4,4'-OxydianilineDetected In Diet Samples

 

II

90-Day Feeding and Reproduction Study In CD® Rats - Male Mean Body Weight

 

III

90-Day Feeding and Reproduction Study In CD® Rats - Male Mean Body Weight Gain

 

IV

90-Day Feeding and Reproduction Study In CD® Rats - Female Mean Body Weight

 

V

90-Day Feeding and Reproduction Study In CD® Rats - Female Mean Body Weight Gain

 

VI

90-Day Feeding Study In Fischer 344 Rats - Male Mean Body Weight

 

VII

90-Day Feeding Study In Fischer 344 Rats - Male Mean Body Weight Gain

 

VIII

90-Day Feeding and Reproduction Study In CD® Rats - Male Mean Daily Diet Consumption

 

IX

90-Day Feeding and Reproduction Study In CD® Rats - Female Mean Daily Diet Consumption

 

X

90-Day Feeding Study In Fischer 344 Rats - Male Mean Daily Diet Consumption

 

XI

90-Day Feeding and Reproduction Study In CD® Rats - Female Mean Daily Diet Consumption

 

XII

90-Day Feeding and Reproduction Study In CD® Rats - Female Mean Food Efficiency

 

XIII

90-Day Feeding Study In Fischer 344 Rats - Male Mean Food Efficiency

 

XIV

90-Day Feeding and Reproduction Study In CD® Rats - Male Mean Daily Intake

 

XV

90-Day Feeding and Reproduction Study In CD® Rats - Female Mean Daily Intake

 

XVI

90-Day Feeding Study In Fischer 344 Rats With Oxydianiline - Male Mean Daily Intake

 

XVII

Clinical Observations Made In Male and Female CD® RatsFed For 90 Days With Diets that Contained 0, 10, 100, or 400 PPM Oxydianiline

 

XVIII

Clinical Observations Made In Male Fischer 344 Rats Fed For 90 Days With Diets That Contained 0, 10, 100, or 400 PPM Oxydianiline

 

XIX

Mean Testes and Final Body Weights of Male Fischer 344 Rats Fed For 90 Days With Diets That Contained 0, 10, 100, or 400 PPM Oxydianiline

 

XX

Mean Relative Testes Weights of Male Fischer 344 Rats Fed For 90 Days With Diets That Contained 0, 10, 100, or 400 PPM Oxydianiline

 

XXI

Mean Testes and Final Body Weights of Male CD® Rats Fed For 173 Days With Diets that Contained 0, 10, 100, or 400 PPM Oxydianiline

 

XXII

Mean Relative Testes Weights of Male CD® Rats Fed For 173 Days With Diets That Contained 0, 10, 100 or 400 PPM Oxydianiline

 

XXIII

Lactation Indices for Litters Produced by CD Rats (Treated Males mated to UntreatedFemales) Fed Diets That Contained 0, 10, 100, or 400 PPM Oxydianiline

 

XXIV

Mean Pup Body Weights Per Litter Produced by CD® Rats (Treated Males mated to Untreated Females) Fed Diets That Contained 0, 10, 100, or 400 PPM Oxydianiline

 

XXV

Reproduction and Lactation Indices for Litters Produced by CD Rats (Untreated Males mated to Treated Females) Fed Diets That Contained 0, 10, 100, or 400 PPM Oxydianiline

 

XXVI

Mean Pup Body Weights Per Litter Produced by CD® Rats(Untreated Males mated to Treated Females) Fed Diets That Contained 0, 10, 100, or 400 PPM Oxydianiline

 

XXVII

Reproduction and Lactation Indices for Litters Produced by CD® Rats (Treated Males mated to Treated Females) Fed Diets That Contained 0, 10, 100, or 400 PPM Oxydianiline

 

XXVIII

Mean Pup Body Weights Per Litter Produced by CD ® Rats (Treated Males mated to Treated Females) Fed Diets That Contained 0, 10, 100,or400PPM Oxydianiline

 

XXIX

Clinical Observations Made In Male and Female CD® Rats Fed Diets That Contained 0, 10, 100,or 400PPM Oxydianiline During the Reproduction Phase

 

Applicant's summary and conclusion

Conclusions:
The "no observable effect level" (NOEL) of 4,4'-oxydianiline for reproductive effects, is considered to be a concentration of 100 ppm (nominal) based on effects on testes, on mean number of pups per litter and on mean female weanling body weight per litter. Taken into account the results from diet analyses, the actual NOEL for reproductive effects was considered to range from 80 to 91 ppm.
Executive summary:

Male CD, female CD and male Fisher 344 rats were fed for six, seven and three months, respectively, diets which contained 0, 10, 100 or 400 ppm of 4,4'-oxydianiline. The objectives of the study were to evaluate the influence of the dietary administration of 4,4'-oxydianiline on the reproduction/lactation function of one generation of CD® rats during the production of two litters of pups.

After approximately 90 days of continuous feeding, all Fischer 344 rats were sacrificed and the reproductive tracts were examined for gross abnormalities. Testes with epididymides were weighed and relative testes weights were calculated. Tissues processed for histopathological examination consisted of testis, epididymis, prostrate (ventral and dorsal), seminal vesicle, coagulating gland, ampullary gland, and urinary bladder.

Following the 90-day feeding study, all surviving CD rats (F0) were used in a 1-generation, 2-litter reproduction study. The rats were mated in the following manner: 10 males from the 10, 100, and 400 ppm groups were mated to untreated females; 10 females from the 10, 100, and 400 ppm groups were mated to untreated males; 10 untreated males were mated to 10 untreated females; and 10 males from the test groups were mated to females from the corresponding test groups.

On day 4 postpartum, the litters (F1A) were culled randomly to 10. Remaining pups were sacrificed and did not receive pathological evaluation. Weanlings (21 days after birth) were weighed, sexed, and sacrificed, and did not receive pathological evaluation.

Approximately 1 week after weaning the last F1A litter, the F0 females were mated again in the same manner as described above, but to different F0 males (from the same group as previous mating) to produce the F1B litters.

Following the last mating, the male F0 CD rats were sacrificed, and testes with epididymides were weighed and the reproductive tract was examined for gross abnormalities.

F1B pups were treated in the same manner as the F1A pups. After weaning of the F1B pups, all F0 females were sacrificed and did not receive pathological evaluation.

During the 90 -day feeding phase, decreases in mean body weights, weight gain and food efficiency values were observed in female CD receiving 400 ppm of 4,4'-oxydianiline. No abnormalities in appearance or behavior were observed in any males or females groups. No mortality occurred during the 90 -day feeding phase. A statistically significant increase in mean aboslute testes weights was observed in Fisher 344 rat fed diets that contains 400 ppm of 4,4'-oxydianiline but with any correlating gross or histomorphological abnormalities. In addition mean absolute and relative testes weights of male CD rats in any test groups were comparable to those of the control group. The biological significance of the decreased testes weights seen in the Fisher 344 rats was considered unclear.

During the reproduction phase, no adverse effect on reproductive function were observed in male CD rats. However in female CD rats, the dietary administration of 400 ppm of 4,4'-oxydianiline adversely influenced reproduction/lactation performance as evidenced by decreased mean number of pups per litter and decreased mean female weanling body weight per litter.

No clinical signs were observed during the reproduction phase of this study in either the F0 parents or in pups.

In conclusion, observations of test item-related testicular effects seen in fisher 344 rats following 90 days of feeding were not confirmed in the reproduction phase.

The "no observable effect level" (NOEL) of 4,4'-oxydianiline for reproductive effects, is considered to be a concentration of 100 ppm (nominal) based on effects on testes, on mean number of pups per litter and on mean female weanling body weight per litter. Taken into account the results from diet analyses, the actual NOEL for reproductive effects was considered to range from 80 to 91 ppm.