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Toxicological information

Toxicity to reproduction

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Administrative data

two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs.
EC Number:
EC Name:
1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs.
Cas Number:
Molecular formula:
Reaction mass of N-phenyl,N'-o-tolyl-phenylene diamine, N,N'-diphenyl-p-phenylene diamine and N,N'-di-o-tolyl-phenylene diamine
Details on test material:
The lot # for the test chemical was 137170393 used for the duration of this study. It was assumed to be 100% pure.

Test animals

Details on test animals or test system and environmental conditions:
Caesarean-originated Sprague-Dawley outbred albino rats were supplied by Charles River Labs, North Carolina. Rats were ~42 days of age (females 100-160 g, males 120-180 g) upon arrival at the laboratory, and at study initiation, the mean body weights of all test females were 184 g, males - ~220g with weight variation maximum of 20%. Animals were quarantined for 7 days prior to initial exposures to test diets. The prebreeding exposure period was 10 weeks in duration.

Diet & drinking water were provided ad libitum. Other than mating periods, animals were housed individually. No fasting of animals was employed in this study. Housing of animals was conducted in a 12/12 hr light/dark cycle with temperature range 68-75 degrees C, and relative humidity of 55 +/- 2.1 %. The in-life study period was conducted beginning June 1996, and ended in March 1997.

Administration / exposure

Route of administration:
oral: feed
corn oil
Details on exposure:
Test chemical was received from Goodyear, and stored at room temperature until used. The material was weighed and added to corn oil with heating up to 90 degrees C to achieve solubization. Proper volumes were then added to achieve targeted concentration in ground rodent diet (NIH-07 Rodent Chow (Ziegler Brothers, PA). Dietary levels of test chemical in the 4 tests groups were: 0, 120, 400, and 1500 ppm while all diets contained 2% corn oil. Diets are made as needed with maximum holding period of 5 weeks. Diets were stored at -15 to -25 degrees C. Feeding jars presented to test animals were renewed with test diets once per week.
Details on mating procedure:
Females in the F0 & F1 generations were paired with males (1:1 non-siblings) until mating was confirmed or until end of mating period (14 days). Females were examined for vaginal sperm and/or copulation plugs for evidence of insemination on successive days until insemination was confirmed. Each female was housed with one male only during study, even when successful mating did not occur. Females were then housed individually following confirmation of mating. Females not mating, not pregnant or not delivering litters were held until scheduled sacrifice for that generation's maternal animals which was following a 3-week lactation period for those rats with live litters.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Feed samples were extracted with toluene by sonication and shaking followed by centrifugation. Supernatants were analyzed by GC using Nitrogen/Phosphorus detector. Test chemical in diets were quantitated using area-under-the-curve calculations based upon standard curves generated from test chemical alone.
Duration of treatment / exposure:
Test animals were exposed as follows: F0 male & females - continuously for 10 weeks prior to mating, and then during the mating period. F0 females & males continued on diet until deliveries occured. Female exposures continued through a 21-day lactation period post delivery. For F1 generation rats, animals were exposed via diet upon their ingestion of maternal diet. Their exposures to both sexes continued through a 10 week period after which housing was arranged for mating of F1 animals. Again, exposures continued for females through the gestation and lactation periods post-deliveries whereas F1 male exposures ended upon F2 deliveries. The F2 generation was not exposed to treated diets except late in the 21-day lactational period.
Frequency of treatment:
Diets, when offered, is available 24 hr/day, 7 days/week.
Details on study schedule:
Rats were approximately 7 weeks of age at commencement of treatment of the F0 generation. Pups from F1 generation were randomly chosen for assignment for mating 21 days post-birth to produce the next generation. F1 rats began a 10-week prebreed exposure period within the range of 26 to 40 days of age. Mating of F1 rats was initiated at 14 - 17 weeks of age.
No. of animals per sex per dose:
30 males, 30 females in each of 3 exposure groups + the negative (diet) controls
Details on study design:
Dose selection was done based upon results from a 4-week dietary study in rats (Iatropoulos, 1994). In that study, the NOEL was 120 ppm while higher doses of 470 ppm to 1900 ppm induced minor effects.
Positive control:


Parental animals: Observations and examinations:
Cage side observations were made & recorded once daily for all gross adverse effects while mortality was monitored twice daily. Body weights were recorded for males weekly throughout study, and for females weekly except during lactation period during which it was on post-natal Days 0, 4, 7, 14, and 21.

Feed consumption was measured weekly for all F0 & F1 animals throught the prebreed & gestational periods but not during mating periods. During lactational period, females were monitored days 0-4, 4-7, 7-14- and 14-21., noting that pup consumption of feed may occur during the latter time period. Actual chemical intakes were estimated from the dietary data. Water consumptions were not measured.
Oestrous cyclicity (parental animals):
Estrous cyclicity was monitored via vaginal cytology during last 3 weeks of 10 week prebreed exposure period for both F0 and F1 rats..
Sperm parameters (parental animals):
Parental male (F0 & F1) reproductive parameters were examined at the times of sacrifice. Testes and epdidymes were weighed. One testis from each animal was frozen at -20 C for enumeration of testicular homogenization-resistant spermatid heads. The opposing testis (left side) was retained in fixative for possible microscopic examinations. One cauda epididymis was immediately removed at sacrifice, weighed, and seminal fluid from the cauda was assessed for sperm number, motility, and morphology. The opposing epididymis was retained for possible microscopic examination. Microscopic exams of testes were made to assess retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing os spermatogenic cells into the lumen.

Males (F1 only) were examined for preputial separation beginning on post-natal day 35.
Litter observations:
On Day 4 following deliveries, each litter was randomly culled to 10 pups with selections made to optimize opportunity for equal numbers of male & female pups. Excess pups were killed and discarded.

Delivered pups in each litter were counted, sexed, weighed individually, and examined grossly at birth at 4, 7, & 14 days after birth, and at weaning on Day 21. Stillbirths, live births, postnatal mortality, gross anomalies, and physical/behavioural abnormalities were recorded. Necropsies were conducted on pups dying during the lactation period.
Postmortem examinations (parental animals):
Male rats in F0 & F1 parental generations were sacrificed at the time females had completed the lactation periods with their litters (post-delivery Day 22). Maternal animals were sacrificed upon completion of weaning of their litters. F1 females were subjected to vaginal lavage to determine the stage of estrus at termination.

Gross necropsy of each adult consisted of examination of external and internal sites including the cranial cavity (external & cut surfaces of the brain & spinal cord), cervical, thoracic, pelvic, and abdominal viscera. Various organs were weighed: ovaries, uteri, testes, epididymides (total & caudal), seminal vesicles, seminal vesicles with coagulating glands & their fluids, prostate, brain, liver, kidneys, adrenal glands and spleen.

The tissues from controls and high dose rats prepared for, and subjected to, microscopic examinations were: ovaries with oviducts, vagina, uteri with cervix, prostate, testis, epididymis, seminal vesicles, kidneys & tissues with gross lesions.
Postmortem examinations (offspring):
Surviving F1 weanling offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age. These animals were subjected to postmortem external examination. In addition, 3 males & 3 females were chosen on day 21 from each litter as were moribund pups for complete necropsies with internal visceral examinations with gross lesions & kidneys retained in fixative. Organ wts were recorded for the thymus, spleen, brain.

Tissues subjected to microscopic examination in weanlings were kidneys in all test groups for F1 & F2 animals,
Reproductive indices:
Those included indices for FEMALES: mating (# females sperm+/# paired females), fertility (# females pregnant/females sperm+), pregnancy (# pregnant females/# males impregnating females). MALES: mating (# male impregnating females/# paired males), fertility (# male siring litters/# males impregnating females), pregnancy (# pregnant females/# males impregnating females).
Offspring viability indices:
Offspring indices: gestational (# females with live litters/# females pregnant), live birth (# live births/# born), survival for 4- (# surviving pups @ 4 days precull/# pups born), 7- (# pups serviving 7 days/live pups @ 4 days postcull), 14- (# pups surviving 14 days/# surviving pups 7 days), & 21-days (# pups surviving 21 days/# pups at 14 days), & lactation (# pups surviving 21 days/live pups at 4 days postcull).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)


F0 Rats

Male rats exhibited an increased incidence (13% in mid-dose, 23% in high-dose 30 rats) of sores often at multiple dermal sites that were treated with antibacterials beginning on Day 55. No other clinical signs were noted in these groups. There was no mortality in males throughout this study.

One female control was euthanized on 17th day of prebreed dosing due to apparent broken hind limb. No clinical signs were associated with test chemical through the mating period in females.

During late gestation (days 23-24), high dose females exhibited increased incidences of palor (13%), piloerection (27%) and vaginal bleeding (17%), the latter likely related to delayed parturition in this group. One mid-dose female was found dead on gestation day 17.

During the lactation period, 2 mid-dose & 2 high-dose females were found dead. 2 high-dose rats were euthanized. 10 females exhibited piloerection while 28% displayed palor.


Fo male body weights were shown to be 10% decreased in high dose animals from the beginning of chemical exposure to the end of the 10-wk prebreed period. BW gains were not different between groups during mating period. Females in the high dose group exhibited the same degree of body wt deficits as males (10%) during prebreed dosing period.

During the gestation period, high dose females gained signficantly less BW during 20 day period than other groups (18% below controls). Feed consumption was not different amongst test groups.

F0 female feed consumption was not significantly different amongst test groups during the lactation period. Body weights did not increase for control, low - or mid-dose females while high dose rats did exhibit increased BWs during this 21-day period, resulting in a return to a mean BW similar to those of controls. Feed consumptions were not significantly different between treatment groups.


F0 males had calculated average chemical intake of 7.6, 25.5, and 98.0 mg/kg-day during the 10-wk prebreed period. Females had intakes of 8.2, 29.2, & 107.5 mg/kg-day during same time period. During gestation, female intakes were 7.4, 25.3 & 95.7 mg/kg-day, and 19.4, 62.3 & 236.5 during lactation.


F0 females exhibited abnormal cycles in 17, 7, 7 and 14% of rats in controls, low, mid, and high dose groups. Cycle lengths were 4.69, 4.50, 4.66, & 4.71 days, respectively.

Effect levels (P0)

open allclose all
Dose descriptor:
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
Dose descriptor:
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
Effect level:
1 500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
reproductive function (oestrous cycle)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
effects observed, treatment-related

Details on results (F1)


F1 found dead pups on PND 0 were 3, 7, 8, and 110 in the control, low, mid & high dose groups. Thereafter, the incidences for dead pups during the lactation period in the F1 generation were .

Effect levels (F1)

Dose descriptor:
Effect level:
1 500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

F0 Reproduction Indices
Starting # M & F pairs 30 30 30 30
Females mated 26 30 26 27
Mating index - %(a) 90 100 87 90
Fertility index - %(b) 92 90 92 93
Gestational index - %(c) 100 96 100 60*
Pregnancy index - %(d) 92 90 92 93
Gestational length - days 22.2 22.4 22.8** 23.5**
*  p<0.01 Fisher exact test
** p<0.01 Dunnett's test
(a) # mated/# paired
(b) # pregnant/# mated
(c) # females with live litters/# pregnant
(d) # pregnant females/# males that mated

F1 Litter Results
Endpoint Controls 120 ppm 400 ppm 1500 ppm
Live litters PND 0 24 26 22 15
# implantation sites/litter 16.9 15.8 15.6 14.5
Total pups/litter PND 0 15.7 14.9 12.3** 12.1**
Live pups/litter PND 0 15.6 14.1 11.9# 7.6**
Live birth index - %(a) 99.2 98.0 97.0 57.5*
4-Day survival - %(b) 98.3 99.0 95.4 83.5
7-Day survival - %(c) 100 100 99.5 100
Lactational index - %(d) 99.6 100 99.5 100
Female pup wt per litter PND 0 6.22 6.63# 6.67** 6.44
Female pup wt per litter PND 21 53.91 58.29 60.37# 55.39
*  p<0.01; Mann-Whitney
** p<0.01 Dunnett's test
#   p<0.05 Dunnett's test
(a) # live pnd 0/# delivered
(b) # live day 4/# live pnd 0
(c) # live day 7/# live pnd 4
(d) # live day 21/# live pnd 4

F1 Reproduction Indices
Endpoint Controls 120 ppm 400 ppm 1500 ppm
Starting # M & F pairs 30 30 30 30
Females mated 26 26 24 26
Mating index - % 87 87 80 87
Fertility index - % 85 88 92 93
Gestational index - % 100 96 91 92
Pregnancy index - % 85 88 92 92
Gestational length - days 22.2 22.8** 23.1** 23.2**
** p<0.01 Dunnett's test

F2 Litter Results
Endpoint Controls 120 ppm 400 ppm 1500 ppm
Live litters PND 0 22 22 20 21
# implantation sites/litter 16.5 16.3 15.1 15.0
Total pups/litter PND 0 15.7 14.5 15.2 13.3
Live pups/litter PND 0 15.6 13.7 13.4 10.8**
Live birth index - %(a) 99.2 91.9 97.2 77.8*
4-Day survival - %(b) 88.6 95.0 98.7 83.1
7-Day survival - %(c) 94.8 100 98.9 91.6
Lactational index - %(d) 94.8 100 97.9 89.5
Female pup wt per litter PND 0 6.17 6.73* 6.82* 6.48@
Female pup wt per litter PND 21 53.2 55.4 58.9** 50.7
*  p<0.01, Mann-Whitney; @ p<0.05
** p<0.01 Dunnett's test

Applicant's summary and conclusion

A 2-generation study was conducted using dietary exposures to rats to the submission substance at 120, 400, & 1500 ppm. Relatively minor toxic effects related to chemical exposure were observed in parental animals including a minimal severity of renal polycystic changes. Reproductive effects included decreases in incidence of live pups/litters in both generations, and a prolongation of gestation in F0 & F1 maternal animals in all dose groups. The NOEL for the study was <120 ppm.
Executive summary:

Dietary exposure to DAPD for 2 generations of rats resulted in parental toxicity at 120, 400, & 1500 ppm. Polycystic kidneys were present in F0 & F1 adults, and in F1 & F2 weanlings in all treatment groups. The renal results for all populations in lowest exposure group (120 ppm) exhibited only minimal polycystic changes. Slight increases were seen in relative liver wts in parental F0 & F1 male high dose rats. Reproductive effects included a decreased gestational index in the F0 generation at the high dose only, and increased gestational lengths in the F0 (mid & high dose) and F1 (all doses) deliveries. The number of total & live births per litter on post-natal day 0 were diminished to a degree in all dose groups of the F1 & F2 generations with statistical significance seen in the mid and high dose F1 litters, and high dose F2 litter.

No reproductive toxicity was observed in males due to chemical exposures. The most pronounced effects in females was delayed parturition which may have been associated with the increased perinatal mortality and decreased live births. This finding has been previously observed for R-59 (N,N'-diphenyl-p-phenylene) in the published literature. The NOEL for this study is <120 ppm dietary level.