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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
publication
Title:
Diverse developmental toxicity of di-n-butyl phthalate in both sexes of rat offspring after maternal exposure during the period from late gestation through lactation
Author:
Kyoung-Youl Lee, Makoto Shibutani, Hironori Takagi, Natsumi Kato, Shu Takigami, Chikako Uneyama, Masao Hirose
Year:
2004
Bibliographic source:
Toxicology 203, 2004, p. 221–238

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Study was performed to evaluate developmental toxicity of di-n-butyl phthalate (DBP) with exposure during the period from late gestation (GD 15) to following lactation (PND 21), maternal rats were given DBP at dietary concentrations of 0, 20, 200, 2000 and 10,000 ppm. 
GLP compliance:
not specified

Test material

Constituent 1
Reference substance name:
di-n-butyl phthalate
IUPAC Name:
di-n-butyl phthalate
Test material form:
not specified
Details on test material:
DBP was purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan; CAS no. 84–74–2, Cat no. P0292, purity >98%)

Test animals

Species:
rat
Strain:
other: pregnant CD®(SD)IGS rats from Charles River Japan Inc. (Kanagawa, Japan) at gestational day 3 (GD 3, the day when vaginal plugs were observed was designated as GD 0).
Details on test animals or test system and environmental conditions:
- housed individually in polycarbonate cages (SK-Clean, 41.5 cm × 26 cm × 17.5 cm in size; CLEA Japan Inc., Tokyo) on wood chip bedding (Soft Chip; Sankyo Lab Service Corp., Tokyo, Japan)
- maintained in an air-conditioned animal room:
- temperature 24 ± 1 ◦C,
- relative humidity 55 ± 5%
- with a 12-h light/dark cycle
They were allowed ad libitum access to feed and tap water.
CRF-1, a regular rodent diet, obtained from Oriental Yeast Co. Ltd. (Tokyo, Japan) was employed as the basal diet for offspring, while dams from GD 3 to PND 21 (the day of delivery was designated as PND 1), received soy-free diet (Oriental Yeast Co. Ltd.), prepared based on the NIH-07 open-formula rodent diet, and with nutritional standards not differing from those of the CRF-1 (supplier’s analysis).

Concentrations of estrogens and phytoestrogens in the soy-free diet were as described previously (Masutomi et al., 2004a).
Briefly, phytoestrogens were below the detection limit (<0.05 mg/100 g diet), except for coumestrol present at 0.3 mg/100 g.

Administration / exposure

Route of administration:
oral: feed
Duration of treatment / exposure:
Immediately after arrival at the testing facility, dams were provided with powdered soy-free diet.
On GD 15, these animals, weighing 320–330 g, were randomized into five groups (6–8 dams/group) and provided with soy-free diet that contained DBP at concentrations of 0, 20, 200, 2000 and 10,000 ppm until PND 21.
Doses / concentrations
Remarks:
Doses / Concentrations:
soy-free diet that contained DBP at concentrations of 0, 20, 200, 2000 and 10,000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
6–8 dams/group

offspring: on PND (post natal day) 3, each litter was culled randomly to give a combination, where possible, of four males and four females.

On PND 21, dosing was terminated, and the offspring were weaned and grouped as follows;
- eight males and eight females (at least one male and one female per litter) per group for prepubertal necropsy;
- eight to ten males and eight females (at least one male and one female per litter) per group for necropsy at PNW (post natal week) 11;
- eight to ten animals of each sex (at least one male and one female per litter) per group for necropsy at PNW 20.
Control animals:
yes
Details on study design:
On PND 2, the numbers, weights and AGDs of the neonates were recorded, and on PND 3, each litter was culled randomly to give a combination, where possible, of four males and four females.
The culled pups were killed by ether anesthesia.
On PND 14, male pups were inspected for the presence and numbers of nipples/areolae. Until weaning at PND 21, body weights of pups were recorded once a week.

On PND 21, dosing was terminated, and the offspring were weaned and grouped as follows;
- eight males and eight females (at least one male and one female per litter) per group for prepubertal necropsy;
- eight to ten males and eight females (at least one male and one female per litter) per group for necropsy at PNW (post natal week) 11;
- eight to ten animals of each sex (at least one male and one female per litter) per group for necropsy at PNW 20.

Diet was changed to CRF-1 at weaning to eliminate possible modifications due to the long-term use of soy-free diet on the development after weaning (Masutomi et al., 2003, 2004a).

Examinations

Maternal examinations:
The body weight and food intake of all dams were recorded at GDs 15 and 20, as well as PNDs 2, 10 and 21.
Fetal examinations:
Prepubertal necropsy was conducted at PND 21 to evaluate weights and histopathology of endocrine-linked organs (see details below).
All female pups were monitored daily for vaginal opening from PND 27 and all male pups were examined for preputial separation from PND 35 until each animal acquired this developmental landmark. The age at the onset of puberty was thus recorded. Estrous cyclicity of females was examined by daily microscopic observation of vaginal smears during PNW 8–11 and PNW 17–20 for at least 21 days from the first day to the end of each period. Classification was into proestrus, estrus and diestrus, when nucleated epithelial cells, cornified epithelial cells or leukocytes, respectively, were contained. ‘Extended diestrus’ or ‘extended estrus’ was concluded when diestrus or estrus continued for at least 4 days (Masutomi et al., 2003, 2004a).

At PNWs 11 and 20, offspring were subjected to organ weight measurement and histopathological examination of endocrine-linked organs.

Male offspring were killed on the first day of each time point.
For female offspring, killing was delayed for up to 4 days after the first day of PNW 11 and PNW 20 until the animal entered the diestrus stage of the estrous cycle.
Statistics:
Data for offspring obtained during the lactation period, that is, body weights on PND 2, AGD and body weight gain, were analyzed using the litter as the experimental unit.
Data for offspring after weaning, as well as the maternal data, were analyzed using the individual animal as the experimental unit.
Differences between groups were evaluated using the following methods.

Numerical data were analyzed for homogeneity of variance using Bartlett’s test:
- When the variance was homogenous among the groups, a one-way analysis of variance (ANOVA) was carried out. If a significant difference was found, the mean value for each treatment group was compared to that of the controls using Dunnett’s test.
- When the variance was heterogeneous based on Bartlett’s test, the Kruskal–Wallis’s H-test was employed to check for differences among the groups. If significant differences were found, a Dunnett-type rank-sum test was performed.

The incidences of nipple retention, histopathological lesions and vaginal cyclicity were statistically compared using the Fisher’s exact probability test.
Severity of histopathological lesions was compared using the Mann–Whitney’s Utest.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
During GDs 15–20, body weight gain of dams was slightly decreased in the 20 and 10,000 ppm dose groups.
Maternal food consumption was not changed by DBP exposure. Also, during the lactation period from PND 2 to PND 21, neither maternal body weight gain nor food consumption was altered. Therefore, levels of maternal daily intake of DBP were concluded to be proportional to the dose.
Duration of pregnancy was also not changed by DBP.

Effect levels (maternal animals)

Dose descriptor:
LOAEL
Effect level:
20 ppm
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Effects on offspring until prepubertal necropsy

Numbers of live offspring were not changed at any dose of DBP. However, the male ratio at birth was slightly reduced at 2000 ppm (43.9 ± 15.7%), and strongly reduced at 10,000 ppm (24.7 ± 4.5%), while the control value tended to be high (65.6 ± 14.2%).

AGD measured on PND 2 was reduced in males at 10,000 ppm, as compared with the control value. Values for males at lower doses and females were not altered.

Body weight at this time point was increased in the 20 ppm-exposed animals of both sexes. On PND 14, retention of nipples/areolae was apparent in males at 10,000 ppm, and a tendency for increase in the incidence was apparent with increase of the dose level

from lower doses.

At prepubertal necropsy, slight, but non-significant reduction of the body weight was observed at 10,000 ppm in both sexes. Increase of the relative weight of the liver was apparent in these animals. In males at this dose, increase of relative weight of the brain and decrease of both absolute (data not shown) and relative weights of the testis were also observed. A tendency for increase in the relative brain weight was similarly noted in females at this dose. Additionally, decrease of absolute testicular weight was observed at 20 ppm (data not shown). Other organs in both sexes did not demonstrate alteration in weights due to DBP.

Efects on the onset of puberty and estrous cyclicity

Regarding preputial separation, early onset was observed at 200 ppm, but this was attributable to the influence of one pup with a markedly earlier onset time than other pups of this group. Males at other doses did not show any fluctuation in the onset time. In

females, on the other hand, slight but non-significant delay in the age of onset of vaginal opening was observed at 10,000 ppm (approximately 1.5 days).

In terms of estrous cyclicity, irregularity judged as extended diestrus was observed in one each out of eight females in the control, 20 and 200 ppm groups during PNWs 8–11. At the 2000 and 10,000 ppm doses, this irregularity was observed in two and four out of eight females, respectively, but the changes were statistically non-significant as compared with the control value. During PNWs 17–20, extended diestrus was observed in three out of 10 females at 2000 ppm (statistically insignificant), while similar change was observed in one case each out of 10 females in the control, 20 and 10,000 ppm groups. In addition, extended estrus was observed in one of eight females at 200 ppm. During both examined periods, all animals not showing extended estrus or extended diestrus retained regular cycle length of 4–5 days.

Organ weight changes at the adult stage

At PNW 11 necropsy, body weights did not significantly fluctuate with the treatment in either sex, while slight, but insignificant increase was observed at 200 and 10,000 ppm in males and 200 ppm in females. In males, slight reduction of the relative weight

of the kidneys was detected at 10,000 ppm. On the other hand, a non-monotonic dose-dependent response was observed in the pituitary weight, where increase of both absolute (data not shown) and relative weights was observed at 200 and 2000 ppm, the relative value also being increased at 20 ppm. In addition, increase of both absolute (data not shown) and relative weights

of the ventral prostate was observed at 200 ppm. In females, a tendency for decrease in the relative pituitary weight was observed with increase of the dose level from 200 ppm, with significant decrease of both absolute (data not shown) and relative values at 10,000 ppm.

Applicant's summary and conclusion

Conclusions:
Reduced spermatocyte development in prepubertal rats and mammary gland changes in adult male rats perinatally (GD 15 to PND 21) exposed to 2 mg DBP/kg bw/day and above via the diet were found. No NOAEL was determined. The LOAEL was determined to be 20 ppm in the maternal diet (1.5–3.0 mg/kg/day. Additionally, anogenital distance was reduced and nipple retention was increased in males at 1000 mg DBP/kg bw/day.
Executive summary:

Study was performed to evaluate developmental toxicity of di-n-butyl phthalate (DBP) with exposure during the period from late gestation (GD 15) to following lactation (PND 21), maternal rats were given DBP at dietary concentrations of 0, 20, 200, 2000 and 10,000 ppm. 

At 10,000 ppm, male offspring showed a decreased neonatal anogenital distance and retention of nipples (PND 14), while females showed a slight non-significant delay in the onset of puberty. At PND 21, reduction of testicular spermatocyte development was evident from 20 ppm, as well as mammary gland changes at low incidence in both sexes. At this time point, population changes of pituitary hormone-immunoreactive cells were observed at 10,000 ppm with a similar pattern of increase in the percentages of luteinizing hormone (LH)-positive and decrease in follicle-stimulating hormone (FSH) and prolactin producing cells in both sexes, effects also being evident on FSH from 200 ppm and LH from 2000 ppm in females. At adult stage necropsy, testicular lesions appeared to be very faint in most cases, but degeneration and atrophy of mammary gland alveoli were observed in males from 20 ppm. Although without clear monotonic dose-dependence, relative pituitary weights were increased with the intermediate doses in males at PNW 11. In females, relative pituitary weights were decreased after 10,000 ppm at PNW 11, and from 200 ppm at PNW 20. The proportion of FSH-positive cells in the pituitaries at PNW 11 was increased in both sexes at 10,000 ppm. Thus, developmental exposure to DBP affected female sexual development involving pituitary function, while in males testicular toxicity was mostly reversible but mammary gland toxicity was persistent
at a dose level as low as 20 ppm (LOAEL).

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