Dicyclohexyl phthalate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 18, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published literature fulfilled basically scientific principles

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

No declaration of guideline compliance in the article, but it seems the method was similar to the OECD guideline 416.

GLP compliance:

no

Remarks:

Publication

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dicyclohexyl phthalate

EC Number:

201-545-9

EC Name:

Dicyclohexyl phthalate

Cas Number:

84-61-7

Molecular formula:

C20H26O4

IUPAC Name:

1,2-dicyclohexyl benzene-1,2-dicarboxylate

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Administration was started at five weeks of age in the F0 parental animals. After quarantine and acclimatization for 5 days, healthy rats were subjected to the study. Animals were reared on basal diet (NIH-07M, CLEA Japan, Inc.) and tap water irradiated by ultraviolet-ray after filtration through a 5 µm filter was given ad libitum using water bottles. They were housed in an animal room controlled with the temperature at 19.0°-25.0 °C, the relative humidity at 35.0%-75.0%, the number of ventilation at about 12 times/hour, and the light and dark cycle at 12 hours (turn on at 7 o’clock and off at 19 o’clock). They were placed in polycarbonate cages with bedding for laboratory animals. Administration to the F0 parental animals was started at five weeks old.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

In the test, the highest dose for the main study set at 6000 ppm, with middle and lowest doses of 1200 and 240 ppm, respectively, by dividing with the common ratio of 5.

The rats received DCHP in the diet. Administration to F0 parental animals was started from the age of five weeks for both males and females; administration to F0 males was continued until necropsy through 10 weeks or more of the pre-mating and mating periods, while administration to F0 females lasted until necropsy through 10 weeks or more of the pre-mating, mating, gestation, and lactation periods until weaning of the F1 offspring (PND 21). Administration to F1 parental animals was started from the time of weaning (three weeks old) and was continued for F1 males until necropsy through 10 weeks or more of the pre-mating and mating periods, and similarly for F1 females through 10 weeks or more of the pre-mating, mating, gestation, and lactation periods until weaning of the F2 offspring (PND 21). Administration to the non-delivering F0/F1 animals continued until necropsy, which was conducted at least 26 days after confirmation of copulation.

Details on mating procedure:

At the age of 15 weeks after the 10-week administration period starting from five weeks of age for the F0 animals, and at the age of 14-15 weeks after the 10 week or longer administration period from three weeks of age in the F1 animals, females were moved to the cages of male partners in the evening, and the males and females cohabited at a sex ratio of 1:1 through the mating period. The existence of a vaginal plug or sperm in the vaginal smear was assessed every morning from the following day. When either was detected, it was judged that copulation had occurred and the day was defined as gestation day (GD) 0. The mating period was limited to two weeks.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

F0 was started from the age of five weeks for both males and females; administration to F0 males was continued until necropsy through 10 weeks or more of the pre-mating and mating periods, while administration to F0 females lasted until necropsy through 10 weeks or more of the pre-mating, mating, gestation, and lactation periods until weaning of the F1 offspring (PND 21).

F1 was started from the time of weaning (three weeks old) and was continued for F1 males until necropsy through 10 weeks or more of the pre-mating and mating periods, and similarly for F1 females through 10 weeks or more of the pre-mating, mating, gestation, and lactation periods until weaning of the F2 offspring (PND 21).

Administration to the non-deliver-ing F0/F1 animals continued until necropsy, which was conducted at least 26 days after confirmation of copulation.

Frequency of treatment:

In the administration period the animals were dosed every day by diet.

Details on study schedule:

- Selection of parents from F1 generation when pups were PND 21.
- Age at mating of the mated animals in the study: [15] weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg diet

Remarks:

nominal in diet
control group

Doses / concentrations 2

Dose / conc.:

240 mg/kg diet

Remarks:

nominal in diet

Doses / concentrations 3

Dose / conc.:

1 200 mg/kg diet

Remarks:

nominal in diet

Doses / concentrations 4

Dose / conc.:

6 000 mg/kg diet

Remarks:

nominal in diet

No. of animals per sex per dose:

Twenty-four male and female rats were used in each group.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: 5

Positive control:

not required.

Examinations

Parental animals: Observations and examinations:

All adult rats were observed daily for clinical signs of toxicity, and body weights and food consumption were recorded weekly. The body weights and food consumption of copulation-confirmed females were measured on GDs 0 (the day when copulation was confirmed), 7, 14, and 20, on lactation days (LDs) 0, 4, 7, 14, and at necropsy after weaning (day 21 of lactation).

Oestrous cyclicity (parental animals):

Vaginal smears were collected from the females everyday in the morning to examine the estrous cycle during the two weeks before mating, starting from 13 weeks of age for F0 parents and from 11 weeks of age for F1 parents, and the mean days of the estrous cycle were calculated. Estrous cycles other than 4 to 6 days were regarded as abnormal.

Sperm parameters (parental animals):

Sperm motility of ten animals of the parental animals in each F0 or F1 group was assessed with a sperm auto-analyzer (HTM-IVOS Ver.10.8). Ten animals of the control and 6000 ppm groups were also randomly selected for counts of the numbers of homogenization resistant spermatids in the testes (spermatid head counts) and numbers of sperm in the cauda epididymal determined with the sperm auto-analyzer (HTM-IVOS Ver.10.8). A statistically significant difference in the numbers of homogenization-resistant spermatids in the testes was evident between the 6000 ppm group and the control in the F1 parents, so that sperm examination was also added for the 240 and 1200 ppm groups.
Furthermore, ten animals each in the control and 6000 ppm groups, smear specimens were prepared and examined for morphologically abnormal sperm to calculate their appearance rate.

Litter observations:

1) Observation and measurements for offspring
The pups were observed for clinical signs from the outside of the cage everyday through the lactation period. When body weights were measured, each was individually and closely observed in the investigator’s hands. The number of the pups per litter was adjusted randomly to eight (four each of the males and females when possible) on PND 4. When the number of the pups per litter was less than eight, they were housed as is. The pups not selected at the adjustment were sacrificed by exposure to carbon dioxide gas and their whole bodies were stored in 10% neutral phosphate buffered formalin solution.
Body weights were measured on PNDs 0, 4, 7, 14, and 21. The anogenital distance (AGD: the distance between the anus and the genital node) was determined on PND 4 following adjustment of the number of pups. The relative AGD value divided by cubic root of the body weight on the measurement day was also calculated. The F1 and F2 pups were observed for the appearance of areolae (or nipples) on PND 14* and on PND 12*, respectively, and appearances rates (incidences) were calculated.

2) Physical development and sexual maturation
The incidence of pinna unfolding was calculated by observation of all offspring from PNDs 2 to 4. Off spring were also observed and examined for the day on which the completion of the following parameters occurred before weaning: upper incisor eruption from PND 6 and eye opening from PND 10. Regarding sexual maturation, preputial separation in the males and vaginal opening in the females employed as parameter, and the F1 animals were individually observed during the period from PND 35 or PND 27 until completion of male or female sexual maturation, respectively, and the body weights were measured on the day of completion.

3) Reflex response tests
All live pups were examined on PND 19 for their pain response, negative geotaxis, air righting reflex, and the pinna reflex, then the positive rates were calculated.

4) Organ weights (weaning)
The brain, thymus and spleen of single F1 and F2 male and female offspring selected from each litter were weighed on PND 21.

Postmortem examinations (parental animals):

All the parental animals sacrificed or found dead were necropsied. Histological examination was conducted for the following organs from all the male and female F0 and F1 parents of the control and the 6000 ppm groups: the brain, pituitary gland, thyroid including parathyroid, liver, kidneys, adrenal glands, spleen, testes, epididymes, seminal vesicles (including coagulating glands), prostate (ventral lobe), ovaries, uterus (including the cervical region), vagina, and mammary glands. Furthermore, histopathological examination was performed for the groups receiving the doses of 240 or 1200 ppm were conducted as follows: for the liver and thyroid in the male and female F0 and F1 parent animals, for the kidneys in male F0 and F1 parents, for testes in male F1 parents, for adrenals in male and female F1 parents and for macroscopic abnormal sites observed were examined. In addition, any animals that died during the course of the study were examined to investigate the cause of death.

Postmortem examinations (offspring):

Excluding the pups that died before selection on PND 4 or the pups not selected on PND 4 (when their numbers were adjusted), all the remaining pups were necropsied when they were sacrificed or found dead.

Statistics:

Data concerning effects on the offspring until their weaning were based on values calculated per litter as the specimen unit. Using weights of bilateral organs, the sums of the left and right organs were employed for statistical analysis.
Metric data were analyzed for homogeneity of variance by Bartlett’s method. When the variance was homogeneous, one-way ANOVA was carried out. When not homogeneous, on the other hand, a Kruskal-Wallis’s test was performed. When a significant inter-group difference was found, Dunnett’s method or a Dunnett type multiple-comparison method were applied. For some examination items, the Kruskal-Wallis test was applied first, and when a significant inter-group difference was found, Dunnett type multiple-comparison method was conducted. Numerical data were analyzed by the Fisher’s exact probability method. The level of statistical significance was basically set at 5%. For clinical signs, necropsy, and histopathological findings, no statistical analyses were performed.

Reproductive indices:

Reproductive capability (mating, fertility, gestation and birth index)

Offspring viability indices:

Daliy chemical intake; Food consumption; Estrous cycle; Reproductive data; Serum hormone levels; Organ Weights

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in clinical signs in the F0 and F1 parental animals of any dose group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The males and females of F0 and F1 parental animals of the 1200 or 6000 ppm demonstrated inhibition of body weight gain. Treatment with DCHP reduced food consumption in the F1 male parents of the 1200 and 6000 ppm and only 6000 ppm of F0 female parents.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The males and females of F0 and F1 parental animals of the 1200 or 6000 ppm demonstrated inhibition of body weight gain. Treatment with DCHP reduced food consumption in the F1 male parents of the 1200 and 6000 ppm and only 6000 ppm of F0 female parents.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination revealed diffuse hypertrophy of hepatocytes in F0 males and females of the 1200 ppm or higher groups, and in F1 males and females of the 6000 ppm group.

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

In the F0 females receiving 6000 ppm, prolongation estrous cycle was observed but this was not evident in the significant elevation of the estrous cycle F1 females.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

No treatment related effects were observed for the sperm of F0, but In the F1 male parents of the 1200 and 6000 ppm groups, significantly reduced values were observed for the numbers of homogenization-resistant spermatids in the testis.

Reproductive performance:

no effects observed

Description (incidence and severity):

No significant differences were observed in any dose groups of F0 and F1 parental animals.

Details on results (P0)

CLINICAL SIGNS, MORTALITY, BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No treatment-related changes were observed in clinical signs in the F0 and F1 parental animals of any dose group. No deaths occurred during the study period in either sex of the F0 parents. Of the F1 parents, one female in the 240 ppm group died during delivery on GD 25, and one female in the 6000 ppm group died on PND 26. In the former (the dam in the 240 ppm group), although her delivery started on GD 22, all the pups died without suckling the milk, and the dam eventually died on GD 25 before all the intrauterine fetuses were completely delivered. In the latter case
(in the 6000 ppm group), no specific changes were noted. It was considered that the mortality was caused by systemic worsening of general condition during the puerperal period or by opportunistic infection. As no similar changes were found in scheduled-sacrificed animals, it was judged that the changes were not related to DCHP.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
In the F0 females receiving 6000 ppm, prolongation estrous cycle was observed but this was not evident in the significant elevation of the estrous cycle F1 females.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
In the F0 male groups treated with the doses up to 6000 ppm, no treatmentrelated effects were observed for the sperm motility rate, number of homogenization-resistant spermatids in the testis, number of sperm in the caudal epididymis, and incidence of morphologically abnormal sperm. In the F1 male parents of the 1200 and 6000 ppm groups, significantly reduced values were observed for the numbers of homogenization-resistant spermatids in the testis. In the F1 male parents of the 6000 ppm group, soft and small sized testes were observed in one animal, and examination for this rat revealed no sperm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant differences were observed in any dose groups of F0 and F1 parental animals for the following indices; number of days required for completion of copulation, number of estrous stages missed until completion of copulation, mating index, fertility index, gestation length, gestation index, birth index and number of implantations. Furthermore, no effects of the substance were apparent in the results obtained during the delivery and lactation periods. The values for the mating and fertility indices showed slight tendencies for decrease in the 6000 ppm group of the F1 parents, and this was considered associated with the testicular changes (soft and/or small size), recognized in three males at necropsy. In the other males of the same group (the F1 6000 ppm group) copulation and resultant pregnancy were normal.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In the 6000 ppm group, significant increase in relative liver weights in F0 parental males and females and of thyroid in F0 parental males was noted. In addition, regarding the corresponding absolute organ weights, tendencies for increase were apparent. Significant lowering of values was observed for both absolute and relative prostatic weights in F1 parental males in the same group. In the 240 and 1200 ppm groups, no obvious changes were observed in either F0 or F1 males or females.

GROSS PATHOLOGY and HISTOPATHOLOGY (PARENTAL ANIMALS)
As necropsy findings, enlargement of the liver was observed in F0 and F1 parental males and females of the 6000 ppm group and in F1 parental females of the 1200 ppm group. Soft small sized testes were observed in F1 males in the 6000 ppm group. On examination of the reproductive organs, in F0 parental males, focal atrophy of seminiferous tubules was noted in one each of the control and 6000 ppm groups, and no difference in the status was observed. In F1 parental males, three animals in the 6000 ppm group showed diffuse atrophy of seminiferous tubules; along with focal atrophy one in the control, two in the 1200 ppm, and six in the 6000 ppm group. Moreover, in the 6000 ppm group, increase in the degree of atrophic testicular changes was revealed. A lack of sperm in the epididymal tubes was observed in the three males with diffusely atrophic seminiferous tubules.
Histopathological examination revealed diffuse hypertrophy of hepatocytes in F0 males and females of the 1200 ppm or higher groups, and in F1 males and females of the 6000 ppm group. Hypertrophy of the thyroid follicular cells was observed in F0 males of the 1200 ppm or higher groups, F0 females of the 6000 ppm group, and F1 males and females of the 6000 ppm group. Hypertrophied follicular epithelial cells appeared to be high-columnar, and follicles lined with such cells containing small amounts of colloid. While the shape of normal follicular antrum was almost circular, that lined by hypertrophic cells appeared to be irregular. Increased hyaline droplets in the renal proximal tubular epithelium were observed in both F0 and F1 parental males in the 6000 ppm group. Marked change (moderate grade) of the increased hyaline droplet accompanied with the appearance of eosinophilic microbodies. Moderately increased hyaline droplets were also observed in one each of the F0 and F1 males of the control and 240 ppm groups, however, increase in the number of the cases with hyaline droplet was shown in the cases with moderate grade in the 6000 ppm group.

OTHER FINDINGS (PARENTAL ANIMALS)
Serum hormone levels:
Changes with statistical significance included elevated testosterone in F0 parental males of the 1200 ppm group and the lowered FSH in F1 parental females in the 240 ppm. However, since changes were not evident at 6000 ppm , they were not considered related to DCHP.

Effect levels (P0)

Key result

Dose descriptor:

dose level:

Effect level:

6 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

Remarks on result:

other: In the F1 and F2 offspring, no changes related to DCHP treatment were found for clinical signs in any dose group.

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

other: In the F1 and F2 offspring, no changes related to DCHP treatment were found for clinical signs in any dose group.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

In the F1 and F2 offspring, no changes related to DCHP treatment were found for clinical signs in any dose group.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No change related to DCHP treatment was observed in any dose group in terms of the number of F1 and F2 pups delivered, viability on PNDs 0, 4, and 21, the results of the reflex response tests and external surface anomalies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

With 6000 ppm, significantly lowered values were found to the body weight gain.

Sexual maturation:

no effects observed

Description (incidence and severity):

It was considered that the test substance did not directly affect sexual maturation of the offspring.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the 6000 ppm group, significant changes in thymus, spleen and brain weights were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Dilatation of the renal pelvis was observed in F1 male or female offspring of the control and treated groups but in only one or two animals and which was not dose-dependent, so they were considered to be unrelated to DCHP.

Histopathological findings:

no effects observed

Description (incidence and severity):

Dilatation of the renal pelvis was observed in F1 male or female offspring of the control and treated groups but in only one or two animals and which was not dose-dependent, so they were considered to be unrelated to DCHP.

Details on results (F1)

VIABILITY AND PHYSICAL DEVELOPMENT(OFFSPRING)
There were no changes in the 240 ppm group. No change related to DCHP treatment was observed in any dose group in terms of the number of F1 and F2 pups delivered, viability on PNDs 0, 4, and 21, the results of the reflex response tests and external surface anomalies. There were no DCHP treatment-related changes observed in terms of the incidence of pinna unfolding, the ages of incisor eruption (upper incisor eruption) and eye opening in both males and females of the F1 and F2 offspring, the ages as well as body weights at vaginal opening in F1 female offspring and at preputial separation in F1 male offspring.

CLINICAL SIGNS AND BODY WEIGHT(OFFSPRING)
In the F1 and F2 offspring, no changes related to DCHP treatment were found for clinical signs in any dose group. With 6000 ppm, significantly lowered values were found as follows: for body weights on PNDs 0-21 in males and females, for body weight gain on PNDs 7-21 in males and PNDs 4-21 in females in the F1 offspring, and for body weights on PND 21 and for body weight gain on PNDs 14-21 in males and females in the F2 offspring.

SEXUAL MATURATION (OFFSPRING)
Although F1 male offspring in the 6000 ppm group showed significantly lowered body weights on the day of preputial separation, the age was not statistically different from that of control, and body weight gain of the group was inhibited throughout after PND 0. Thus, it was considered that the test substance did not directly affect sexual maturation of the offspring.

ORGAN WEIGHTS (OFFSPRING)
In the 6000 ppm group, significant changes in thymus, spleen and brain weights were observed. These changes, however, were found only in either absolute or relative weights, and some showed contrary results between the two. Therefore, it was considered that the changes were attributable to inhibition of body weight gain, which was related to administration of DCHP. In the groups receiving doses of 1200 ppm or less, no test substance related changes were observed.

GROSS PATHOLOGY AND HISTOPATHOLOGY(OFFSPRING)
Dilatation of the renal pelvis was observed in F1 male or female offspring of the control and treated groups. However, these changes appeared in only one or two animals and were not dose-dependent, so they were considered to be unrelated to DCHP. In addition, no abnormalities were observed in the one F2 offspring which died during the lactational period.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

240 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: (16-21 mg/kg bw/d) based on decreased sperm head counts and focal seminiferous tubule atrophy in F1 males at 1200 ppm (80-107 mg/kg bw/d). There were no effects on reproductive endpoints such as fertility, mating, gestation and birth index.

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

240 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1. Daliy chemical intake.                                                        Unit : mg/kg

DCHP (ppm)			0 (Control)	240	1200	6000
F0 males	Total study period^b		-	15.88 ± 1.07^a	79.57 ± 3.32	401.8 ± 15.6
F0 females	Total study period^c		-	20.8	104.19	510.7
	Pre-mating^d		-	17.70 ± 0.95	88.83 ± 6.39	434.6 ± 29.6
	Gestation^e		-	14.30 ± 0.88	69.77 ± 4.04	349.0 ± 15.8
	Lactation^f		-	37.92 ± 2.50	191.6 ± 12.3	932.8 ± 58.2
F1 males	Total study period		-	17.84 ± 0.86	89.89 ± 5.01	457.4 ± 17.3
F1 females	Total study period		-	20.95	107.15	534.2
	Pre-mating		-	19.27 ± 1.36	98.88 ±8.34	483.0 ± 25.7
	Gestation		-	14.11 ± 0.98	72.41 ± 4.18	350.9 ± 19.4
	Lactation		-	33.70 ± 2.27	170.6 ± 14.0	896.7 ± 63.2

a: Mean ± SD.

b: During the period of 10 weeks.

c: The mean daily intake for the study (pre-mating + gestation + lactation periods).

d: Pre-mating period (10 weeks).

e: Gestation length (Days 21-23).

f: Lactation period (Days 0-21). Table 2. Food consumption                                                               Unit : g/animal/day

DCHP (ppm)			0 (Control)	240	1200	6000
F0 males
	Pre-mating^b
	Day 7		19.0 ± 1.3^a	19.6 ± 0.8	19.2 ± 1.0	19.2 ± 1.3
	Day 70		23.0 ± 1.9	23.3 ± 1.2	23.1 ± 1.6	23.1 ± 1.7
F0 females	Pre-mating
	Day 7		14.8 ± 0.9	15.1 ± 1.0	15.0 ± 0.7	14.7 ± 1.2
	Day 49		16.8 ± 1.2	16.7 ± 1.0	16.9 ± 1.7	15.5 ± 1.2*
	Day 70		15.8 ± 0.7	15.8 ± 1.1	15.1 ± 1.2	14.9 ± 0.7
	Gestation^c
	GD 7		20.2 ± 1.9	20.1 ± 1.7	18.9 ± 1.8	18.0 ± 1.7**
	GD 14		22.0 ± 2.6	21.2 ± 2.0	20.4 ± 1.5*	19.4 ± 1.6**
	GD 20		21.3 ± 2.0	21.3 ± 2.4	20.7 ± 1.9	20.8 ± 1.9
	Lactation^d
	LD 4		26.6 ± 4.0	29.3 ± 4.6	30.2 ± 4.6*	28.4 ± 2.4
	LD 21		66.2 ± 4.3	68.1 ± 4.3	69.8 ± 5.5*	64.2 ± 5.1
F1 males	Pre-mating
	PND 28		11.7 ± 0.8	11.9 ± 1.2	11.4 ± 0.8	10.2 ± 1.2**
	PND 56		27.2 ± 1.4	27.1 ± 2.0	26.0 ± 1.2	24.6 ± 1.4**
	PND 91		27.2 ± 1.6	26.8 ± 2.3	25.1 ± 0.7*	24.9 ± 1.4*
F1 females	Pre-mating
	PND 28		10.2 ± 1.1	10.9 ± 1.1	10.6 ± 1.1	9.5 ± 1.1
	PND 56		16.9 ± 1.4	17.9 ± 2.3	17.1 ± 1.9	17.5 ± 2.8
	PND 91		17.0 ± 1.2	18.3 ± 1.8	17.8 ± 0.9	17.0 ± 1.2
	Gestation
	GD 7		21.3 ± 2.2	21.7 ± 2.4	21.4 ± 2.0	20.0 ± 2.1
	GD 14		22.9 ± 3.1	23.5 ± 2.3	23.5 ± 1.9	21.2 ± 2.0
	GD 20		23.2 ± 2.3	23.6 ± 2.4	23.5 ± 2.7	22.4 ± 2.0
	Lactation
	LD 4		29.0 ± 3.3	28.9 ± 3.9	28.4 ± 4.3	29.1 ± 3.7
	LD 21		71.6 ± 5.5	69.6 ± 3.7	70.6 ± 5.1	69.4 ± 4.2

a: Mean±SD.

b: Pre-mating period (10 weeks).

c: Gestation length (Days 21-23).

d: Lactation period (Days 0-21).

GD: Gestation day.

LD: Lactation day.

PND: Postnatal day.

*: Significantly different from the control value, *, p<0.05; **, p<0.01.

Table 3. Estrous cycle

DCHP (ppm)			0 (Control)	240	1200	6000
F0 females
No. of females examined			24	24	24	24
Mean estrous cycle (days)			4.04 ± 0.14^a	4.06 ± 0.22	4.00 ± 0.00	4.25 ± 0.42*
No. of females with abnormal estrous cycles			0/24	0/24	0/24	0/24
F1 females
No. of females examined			19	21	19	21
Mean estrous cycle (days)			4.18 ± 0.38	4.14 ± 0.32	4.21 ± 0.38	4.33 ± 0.46
No. of females with abnormal estrous cycles^b			1/20	2/23	0/20	0/21

a: Mean ± SD.

b: Number of the females with abnormal estrous cycle / number of the females examined.

*: Significantly different from the control value, *, p<0.05; **, p<0.01.

Table 4. Reproductive data.

DCHP (ppm)	0 (Control)	240	1200	6000
F0 parents / F1 offspring
No. of pairs	24	24	24	24
No. of days until copulation (days)^b	2.3 ± 1.3^a	2.0 ± 1.4	2.5 ± 1.1	2.1 ± 1.2
Mating index (%)^c	91.7	95.8	91.7	95.8
Fertility index (%)^d	90.9	100.0	90.9	95.7
Gestation length (days)^e	21.9 ± 0.4	22.0 ± 0.6	22.1 ± 0.5	21.9 ± 0.3
Gestation index (%)^f	100.0	100.0	100.0	100.0
Birth index (%)^g	95.85 ± 4.68	94.35 ± 6.63	94.73 ± 10.01	92.55 ± 7.15
No. of implantation sites	14.4 ± 2.5	14.4 ± 2.4	14.6 ± 2.2	15.0 ± 1.4
Total No. of offspring at birth	13.8 ± 2.6	13.9 ± 2.6	13.9 ± 2.2	14.0 ± 1.6
No. of offspring born alive	13.8 ± 2.6	13.7 ± 2.8	13.8 ± 2.2	13.8 ± 1.5
Sex ratio^h	0.49	0.50	0.49	0.52
Body weights of male offspring
Day 0	6.8 ± 0.6	6.8 ± 0.5	6.8 ± 0.4	6.5 ± 0.4*
Day 4	11.3 ± 1.2	11.4 ± 1.2	11.5 ± 1.2	10.4 ± 1.0*
Day 7	18.5 ± 1.4	18.7 ± 1.6	18.3 ± 1.6	16.7 ± 1.3**
Day 14	37.0 ± 2.4	37.2 ± 2.6	36.8 ± 2.1	33.4 ± 2.0**
Day 21	62.2 ± 4.5	61.9 ± 4.8	62.6 ± 4.6	55.0 ± 3.8**
Body weights of female offspring
Day 0	6.5 ± 0.5	6.4 ± 0.5	6.4 ± 0.5	6.1 ± 0.4*
Day 4	10.9 ± 1.1	11.1 ± 1.2	11.0 ± 1.2	9.8 ± 1.1**
Day 7	17.8 ± 1.3	18.0 ± 1.7	17.5 ± 1.7	15.9 ± 1.5**
Day 14	35.9 ± 2.1	36.2 ± 3.0	35.5 ± 2.3	32.0 ± 2.0**
Day 21	59.2 ± 3.7	59.6 ± 4.9	59.4 ± 4.7	52.8 ± 3.2**
F1 parents / F2 offspring
No. of pairs	20	23	20	21
No. of days until copulation (days)	3.2 ± 1.2	2.5 ± 1.5	1.9 ± 1.2**	2.9 ± 2.3
Mating index (%)	95.0	100.0	100.0	90.5
Fertility index (%)	100.0	95.7	95.0	89.5
Gestation length (days)	21.9 ± 0.2	21.9 ± 0.4	21.7 ± 0.5	22.0 ± 0.5
Gestation index (%)	100.0	100.0	100.0	100.0
Birth index (%)	90.91 ± 6.11	92.85 ± 6.55	94.46 ± 6.66	93.55 ± 8.22
No. of implantation sites	15.9 ± 2.1	15.5 ± 1.6	14.9 ± 2.6	14.3 ± 2.9
Total No. of offspring at birth	14.6 ± 2.2	14.7 ± 1.6	14.2 ± 2.6	13.9 ± 2.9
No. of offspring born alive	14.5 ± 2.3	14.4 ± 1.5	14.1 ± 2.6	13.3 ± 2.5
Sex ratio	0.51	0.50	0.48	0.51
Body weights of male offspring
Day 0	6.8 ± 0.4	6.6 ± 0.5	6.5 ± 0.5	6.6 ± 0.6
Day 4	11.1 ± 0.9	11.0 ± 1.0	10.9 ± 1.0	11.1 ± 1.8
Day 7	18.4 ± 1.3	18.1 ± 1.5	17.8 ± 1.6	18.3 ± 2.2
Day 14	38.5 ± 2.2	37.8 ± 2.5	37.3 ± 2.7	36.2 ± 2.9
Day 21	64.9 ± 4.2	62.8 ± 4.2	62.8 ± 5.0	59.2 ± 5.0**
Body weights of female offspring
Day 0	6.4 ± 0.4	6.2 ± 0.4	6.2 ± 0.5	6.2 ± 0.6
Day 4	10.6 ± 0.7	10.5 ± 0.8	10.4 ± 1.2	10.6 ± 1.5
Day 7	17.6 ± 1.0	17.2 ± 1.3	16.9 ± 1.9	17.5 ± 1.9
Day 14	37.3 ± 2.1	36.3 ± 2.2	35.8 ± 2.7	35.0 ± 2.6
Day 21	61.7 ± 3.7	59.3 ± 3.5	59.2 ± 4.4	56.6 ± 4.3**

a: Mean±SD.

b: No. of days until copulation (days) = Number of days from the start of the mating until the day of copulation confirmed.

c: Mating index (%) = (Number of males and females showing evidence of copulation /

number of males and females used for mating)×100.

d: Fertility index (%) = (Number of pregnant females / number of copulated females)×100.

e: Gestation length (days) = Number of days from GD 0 till the delivery day.

f: Gestation index (%) = (Number of females with normal delivery / number of pregnant females)×100.

g: Birth index (%) = (Number of live pups delivered / number of implantations)×100.

h: Sex ratio = Total number of male pups delivered / total number of pups delivered.

*: Significantly different from the control value, *, p<0.05; **, p<0.01.

Table 5. Data from sperm parameters and hormone levels.

DCHP (ppm)	0 (Control)	240	1200	6000
F0 parents
Sperm examination
No. of males examined	10	10	10	10
Sperm motility (%)	90.7 ± 4.81^a	89.1 ±5.95	87.9 ±6.45	87.4 ± 7.71
Homogenization-resistant spermatids (× 106/g)	110.6 ± 9.02	−	−	104.8 ± 9.34
(spermatid head counts in the testis)
Sperm count (× 106/g), (cauda epididymal)	766.5 ± 102.77	−	−	745.0 ± 87.98
Abnormal sperm (%)	0.5 ± 0.47	−	−	1.0 ± 0.96
Tailless sperm (%)	0.6 ± 0.70	−	−	1.2 ± 1.23
Hormone levels
No. of males examined	6	6	6	6
Testosterone (ng/mL)	1.92 ± 0.75	2.59 ±0.45	3.45 ±  0.74*	2.04 ± 0.87
FSH (ng/mL)	9.88 ± 1.05	10.57 ±1.54	9.67 ±  1.05	9.60 ± 1.37
LH (ng/mL)	1.82 ± 0.17	1.88 ±0.13	1.88 ±  0.20	1.97 ± 0.23
No. of females examined	6	6	6	6
FSH (ng/mL)	6.47 ± 0.69	5.99 ±1.04	6.71 ±  0.68	6.27 ± 0.67
LH (ng/mL)	2.02 ± 0.25	1.85 ±0.31	1.92 ±  0.20	2.02 ± 0.09
Estradiol (pg/mL)	72.85 ± 23.06	72.22 ±24.30	72.25 ± 17.60	81.25 ± 13.76
F1 parents
Sperm examination
No. of males examined	10	10	10	10
Sperm motility (%)	90.7 ± 4.45	92.8 ±3.55	88.7 ±  6.57	80.7 ± 28.66
Homogenization-resistant spermatids (× 106/g)	104.0 ± 12.66	93.4 ±10.27	88.6 ±  10.32*	79.2 ± 30.29**
(spermatid head counts in the testis)
Sperm count (× 106/g), (cauda epididymal)	728.4 ± 88.07	−	−	599.7 ± 219.22
Abnormal sperm (%)	0.2 ± 0.26	−	−	0.3 ± 0.36
Tailless sperm (%)	0.6 ± 0.70	−	−	0.8 ± 1.09
Hormone levels
No. of males examined	6	6	6	6
Testosterone (ng/mL)	2.05 ± 0.75	2.38 ±1.05	2.74 ±  1.24	2.28 ± 1.28
FSH (ng/mL)	8.98 ± 1.32	10.41 ±2.96	10.08 ±  1.40	10.87 ± 1.62
LH (ng/mL)	2.11 ± 0.24	2.00 ±0.24	2.03 ±  0.29	1.81 ± 0.48
No. of females examined	5	6	6	6
FSH (ng/mL)	8.80 ± 3.10	5.50 ±0.65*	6.27 ±  0.94	5.96 ± 0.82
LH (ng/mL)	1.58 ± 0.90	1.99 ±0.35	1.99 ±  0.14	1.80 ± 0.90
Estradiol (pg/mL)	51.28 ± 38.86	82.53 ±13.24	59.25 ±  15.13	85.80 ± 20.27

a: Mean ± SD.

*: Significantly different from the control value, *, p<0.05; **, p<0.01.

−: Not examined.

Table 6. Organ weights for F0/F1 adults.

DCHP (ppm)	0 (Control)	240	1200	6000
F0 adults
No. of male F0 adults examined	24	24	24	24
Final body weight (g)	510.5 ± 50.4^a	503.6 ± 42.5	500.4 ± 28.0	496.7 ± 37.1
Thyroid (L), (mg)	11.33 ± 2.65^b	12.48 ± 2.47	11.99 ± 2.11	14.37 ± 4.02**
	2.23 ± 0.51^c	2.48 ± 0.46	2.40 ± 0.43	2.91 ± 0.91**
Thyroid (R), (mg)	11.74 ± 2.54	11.80 ± 2.14	12.31 ± 3.01	12.63 ± 2.79
	2.33 ± 0.51	2.35 ± 0.42	2.46 ± 0.58	2.54 ± 0.50
Liver (g)	15.042 ± 1.911	14.620 ± 1.960	14.596 ± 1.183	18.157 ± 1.730**
	2.944 ± 0.203	2.898 ± 0.229	2.917 ± 0.177	3.658 ± 0.252**
Prostate (g)	0.712 ± 0.144	0.640 ± 0.140	0.642 ± 0.173	0.657 ± 0.155
	0.141 ± 0.032	0.127 ± 0.024	0.128 ± 0.034	0.134 ± 0.039
No. of female F0 adults examined	20	23	20	22
Final body weight (g)	322.1 ± 19.7	311.9 ± 16.6	306.5 ± 18.2*	295.3 ± 18.8**
Thyroid (L), (mg)	10.08 ± 2.15	9.74 ± 1.59	9.91 ± 1.80	11.35 ± 1.39
	3.13 ± 0.68	3.12 ± 0.49	3.23 ± 0.57	3.87 ± 0.54**
Thyroid (R), (mg)	11.13 ± 2.46	11.14 ± 1.90	10.78 ± 2.16	11.89 ± 2.18
	3.54 ± 0.81	3.58 ± 0.59	3.54 ± 0.69	4.06 ± 0.83*
Liver (g)	10.233 ± 0.994	9.907 ± 0.772	10.298 ± 0.824	11.157 ± 0.995**
	3.174 ± 0.168	3.176 ± 0.171	3.362 ± 0.190*	3.779 ± 0.251**
F1 adults
No. of male F1 adults examined	20	23	20	21
Final body weight (g)	624.9 ± 48.9	603.4 ± 54.0	564.7 ± 42.0**	552.5 ± 30.5**
Thyroid (L), (mg)	15.11 ± 2.23	13.55 ± 3.30	14.24 ± 4.41	15.06 ± 2.77
	2.43 ± 0.46	2.28 ± 0.65	2.52 ± 0.72	2.72 ± 0.48
Thyroid (R), (mg)	16.17 ± 3.34	14.24 ± 3.40	13.61 ± 2.44*	15.66 ± 2.68
	2.62 ± 0.62	2.38 ± 0.67	2.42 ± 0.49	2.84 ± 0.46
Liver (g)	18.822 ± 2.402	17.374 ± 2.137	16.609 ± 1.691**	18.953 ± 2.371
	3.004 ± 0.210	2.877 ± 0.195	2.943 ± 0.238	3.423 ± 0.314**
Prostate (g)	0.707 ± 0.152	0.575 ± 0.133**	0.593 ± 0.149*	0.508 ± 0.118**
	0.114 ± 0.027	0.096 ± 0.020	0.106 ± 0.029	0.092 ± 0.024*
No. of female F1 adults examined	19	22	19	17
Final body weight (g)	337.4 ± 27.8	338.7 ± 23.9	330.8 ± 18.8	320.7 ± 22.7
Thyroid (L), (mg)	12.32 ± 3.48	11.35 ± 1.91	12.07 ± 2.30	11.79 ± 2.55
	3.66 ± 1.05	3.36 ± 0.62	3.66 ± 0.74	3.69 ± 0.85
Thyroid (R), (mg)	14.46 ± 2.92	12.47 ± 2.53	13.33 ± 2.79	13.71 ± 3.16
	4.31 ± 0.87	3.69 ± 0.80	4.05 ± 0.91	4.28 ± 1.05
Liver (g)	12.772 ± 1.844	12.326 ± 1.459	12.907 ± 1.697	14.102 ± 1.918
	3.784 ± 0.455	3.640 ± 0.360	3.902 ± 0.467	4.385 ± 0.393**

a: Mean ± SD.

b: Absolute organ weight.

c: Relative organ weight.

*: Significantly different from the control value, *, p<0.05; **, p<0.01.

Table 7. Histopathological findings for F0/F1 adults.

DCHP (ppm)		Male				Female
		0 (Control)	240	1200	6000	0 (Control)	240	1200	6000
F0 adults
No. of F0 adults examined		24	24	24	24	24	24	24	24
Liver
Hypertrophy, hepatocytes, diffuse	+	0	0	4	16	0	0	3	12
Thyroid
Hypertrophy, follicular cells	+	0	0	3	7	0	0	0	6
Kidney
Hyaline droplet, tubular epithelium, proximal	+	23	22	23	9	0	−	0/1^a	0
	++	1	1	0	15	0	−	0/1	0
F1 adults
No. of F1 adults examined		20	23	20	22	20	23	20	22
Liver
Hypertrophy, hepatocytes, diffuse	+	0	0	0	14	0	0	0	9
Thyroid
Hypertrophy, follicular cells	+	0	0	0	7	0	0	0	6
Kidney
Hyaline droplet, tubular epithelium, proximal	+	19	22	20	14	0	−	−	0
	++	1	1	0	8	0	−	−	0
Testis
Atrophy, seminiferous tubules, diffuse	+++	0	0	0	3
Atrophy, seminiferous tubules, focal	+	1	0	2	6

+, Slight; ++, Moderate; +++, Severe.

a: Number of animals affected / number of the animal examined.

−: Not examined.

Table 8. Anogenital distance and nipple development

DCHP (ppm)		0 (Control)	240	1200	6000
F1 pups
No. of males examined		20	23	20	22
AGD^b(mm)^c		4.683 ± 0.522^a	4.860 ± 0.491	4.757 ± 0.448	4.373 ± 0.354**
AGD/BW^d		2.171 ± 0.216	2.162 ± 0.213	2.107 ± 0.148	2.003 ± 0.151*
Nipple development (%)^e	0	100.0	100.0	100.0	83.3**
	1	0.0	0.0	0.0	16.1**
	2	0.0	0.0	0.0	0.0
No. of females examined		20	23	20	22
AGD (mm)		2.237 ± 0.297	2.355 ± 0.249	2.323 ± 0.310	2.226 ± 0.150
AGD/BW		1.013 ± 0.141	1.059 ± 0.117	1.045 ± 0.114	1.042 ± 0.075
Nipple development (%)	0	100.0	100.0	100.0	100.0
	1	0.0	0.0	0.0	0.0
	2	0.0	0.0	0.0	0.0
F2 pups
No. of males examined		19	22	19	17
AGD (mm)		4.618 ± 0.314	4.494 ± 0.300	4.281 ± 0.365**	4.191 ± 0.387**
AGD/BW		2.072 ± 0.152	2.020 ± 0.125	1.932 ± 0.158**	1.882 ± 0.129**
Nipple development (%)	0	100.0	100.0	81.6	36.8**
	1	0.0	0.0	18.4	63.2**
	2	0.0	0.0	0.0	0.0
No. of females examined		19	22	19	17
AGD (mm)		2.066 ± 0.157	2.125 ± 0.161	2.060 ± 0.169	2.188 ± 0.236
AGD/BW		0.943 ± 0.072	0.972 ± 0.063	0.946 ± 0.062	0.998 ± 0.075
Nipple development (%)	0	100.0	100.0	100.0	100.0
	1	0.0	0.0	0.0	0.0
	2	0.0	0.0	0.0	0.0

a: Mean ± SD.

b: AGD was measured on PND (postnatal day) 4.

c: Distance between the anus and genital node (mm).

d: The relative AGD value divided by cubic root of the body weight on the measurement day was also calculated.

e: Number of litters with abnormal offspring / number of litters examined × 100.

*: Significantly different from the control value, *, p<0.05; **, p<0.01.

Table 9. Viability indices and parameter for physical development and sexual maturation

DCHP (ppm)	0 (Control)	240	1200	6000
F1 pups
Viability index (%)
Day 0 ^b	100.00 ± 0.00 ^a	98.08 ± 3.87	99.25 ± 2.37	98.78 ± 2.67
Day 4 ^c	98.93 ± 2.70	99.09 ± 2.42	99.65 ± 1.59	98.86 ± 5.33
Day 21 ^d	100.00 ± 0.00	100.00 ± 0.00	100.00 ± 0.00	100.00 ± 0.00
Physical development
Pinna unfolding (%)
No. of males examined	20	23	20	22
Day 2	21.2 ± 27.7	34.2 ± 39.0	37.2 ± 39.1	22.1 ± 24.0
Day 3	87.1 ± 32.4	86.6 ± 30.8	96.7 ± 14.9	92.0 ± 26.0
Day 4	99.3 ± 3.2	98.9 ± 3.9	100.0 ± 0.0	100.0 ± 0.0
No. of females examined	20	23	20	22
Day 2	31.1 ± 33.9	33.6 ± 37.8	44.3 ± 39.5	20.3 ± 23.0
Day 3	87.9 ± 30.1	89.5 ± 27.7	93.8 ± 22.8	92.7 ± 24.3
Day 4	100.0 ± 0.0	100.0 ± 0.0	100.0 ± 0.0	100.0 ± 0.0
Incisor Eruption
No. of males examined	20	23	20	22
Age (day) ^e	10.7 ± 0.6	10.8 ± 0.9	10.4 ± 0.8	10.5 ± 0.8
No. of females examined	20	23	20	22
Age (day)	10.8 ± 0.8	10.8 ± 0.9	10.4 ± 0.8	10.5 ± 0.9
Eye opening
No. of males examined	20	23	20	22
Age (day)	14.0 ± 0.6	13.8 ± 0.7	13.9 ± 0.6	14.1 ± 0.4
No. of females examined	20	23	20	22
Age (day)	13.8 ± 0.6	13.6 ± 0.8	13.6 ± 0.5	13.9 ± 0.5
Sexual maturation
Preputial separation (Male)	20	23	20	22
Age (day)	40.6 ± 1.0	40.6 ± 1.2	40.0 ± 0.9	41.4 ± 1.6
Body weight (g) ^f	225.3 ± 17.3	225.1 ± 12.5	218.9 ± 15.4	212.5 ± 13.8*
Vaginal opening (Female)	20	23	20	21
Age (day)	32.6 ± 1.4	31.7 ± 1.7	32.0 ± 1.5	32.5 ± 1.6
Body weight (g)	122.0 ± 11.0	121.1 ± 10.8	118.0 ± 12.9	116.7 ± 8.3
F2 pups
Viability index (%)
Day 0	98.89 ± 2.63	98.30 ± 3.94	99.30 ± 2.13	96.45 ± 7.29
Day 4	98.91 ± 2.60	97.85 ± 3.25	96.39 ± 5.27	97.89 ± 3.38
Day 21	99.34 ± 2.87	100.00 ± 0.00	100.00 ± 0.00	100.00 ± 0.00
Physical development
Pinna unfolding (%)
No. of males examined	19	22	19	17
Day 2	16.1 ± 23.4	9.3 ± 20.5	16.8 ± 30.8	25.7 ± 36.5
Day 3	91.3 ± 24.7	87.8 ± 31.0	75.4 ± 42.5	87.6 ± 29.1
Day 4	100.0 ± 0.0	100.0 ± 0.0	100.0 ± 0.0	100.0 ± 0.0
No. of females examined	19	22	19	17
Day 2	29.8 ± 29.2	12.4 ± 19.3	19.8 ± 31.3	26.8 ± 35.1
Day 3	93.0 ± 23.8	86.1 ± 32.4	76.7 ± 39.1	88.0 ± 28.9
Day 4	100.0 ± 0.0	100.0 ± 0.0	99.5 ± 2.3	99.2 ± 3.5
Incisor Eruption
No. of males examined	19	22	19	17
Age (day)	11.0 ±  0.8	10.7 ±  0.8	11.1 ±  0.7	10.7 ±  0.9
No. of females examined	19	22	19	17
Age (day)	11.0 ±  0.9	10.7 ±  0.9	11.3 ±  0.8	10.8 ±  0.8
Eye opening
No. of males examined	19	22	19	17
Age (day)	13.9 ±  0.5	13.9 ±  0.6	14.2 ±  0.5	14.0 ±  0.6
No. of females examined	19	22	19	17
Age (day)	13.8 ±  0.6	13.9 ±  0.5	14.0 ±  0.6	13.8 ±  0.7

a: Mean ± SD.

b: Viability on PND 0 (survival rate at delivery)(%) = (Number of live pups on PND 0 / total number of pups delivered) × 100.

c: Viability on PND 4 (the survival rate on day 4)(%) = (Number of live pups on PND 4 / number of live pups on PND 0) × 100.

d: Viability rate on PND 21 (weaning rate)(%) = (Number of live pups on PND 21 / number of live pups on PND 4) × 100.

e: Day of completion.

f: Body weight completion day.

*: Significantly different from the control value, *, p<0.05; **, p<0.01.

Table 10. Organ weights for F1/F2 weanlings

DCHP (ppm)	0 (Control)	240	1200	6000
F1 offspring
No. of male F1 weanlings examined	20	22	20	22
Final body weight (g)	62.49 ± 4.64^a	62.44 ± 5.43	61.71 ± 4.65	55.20 ± 3.65**
Brain (g)	1.517 ± 0.054^b	1.521 ± 0.055	1.530 ± 0.042	1.497 ± 0.040
	2.438 ± 0.165^c	2.452 ± 0.207	2.492 ± 0.176	2.721 ± 0.172**
Thymus (mg)	270.10 ± 39.28	262.25 ± 37.58	261.49 ± 34.82	233.07 ± 28.86**
	431.51 ± 47.84	420.47 ± 51.56	424.50 ± 53.53	422.45 ± 46.58
Spleen (g)	0.299 ± 0.053	0.325 ± 0.056	0.321 ± 0.062	0.249 ± 0.039**
	0.477 ± 0.068	0.519 ± 0.069	0.518 ± 0.078	0.451 ± 0.060
No. of female F1 weanlings examined	20	23	20	22
Final body weight (g)	58.86 ± 3.93	59.55 ± 5.39	59.78 ± 5.17	52.27 ± 3.07**
Brain (g)	1.486 ± 0.040	1.468 ± 0.041	1.477 ± 0.051	1.449 ± 0.025**
	2.535 ± 0.177	2.483 ± 0.213	2.486 ± 0.196	2.780 ± 0.152**
Thymus (mg)	265.10 ± 44.49	266.00 ± 38.25	270.11 ± 39.65	239.18 ± 36.12
	449.42 ± 62.19	448.58 ± 66.24	451.39 ± 49.11	456.53 ± 53.44
Spleen (g)	0.286 ± 0.056	0.308 ± 0.063	0.298 ± 0.044	0.247 ± 0.033**
	0.485 ± 0.078	0.517 ± 0.089	0.499 ± 0.060	0.472 ± 0.056
F2 offspring
No. of male F1 weanlings examined	19	22	19	17
Final body weight (g)	66.36 ± 3.86	62.75 ± 5.30	62.79 ± 6.23	59.93 ± 6.48**
Brain (g)	1.515 ± 0.048	1.506 ± 0.059	1.500 ± 0.060	1.525 ± 0.081
	2.289 ± 0.121	2.411 ± 0.151	2.407 ± 0.222	2.567 ± 0.255**
Thymus (mg)	285.17 ± 53.26	251.79 ± 34.70	263.69 ± 46.91	248.24 ± 50.07
	428.79 ± 70.03	401.42 ± 45.16	418.70 ± 53.77	412.00 ± 51.37
Spleen (g)	0.357 ± 0.066	0.337 ± 0.051	0.316 ± 0.055	0.295 ± 0.066**
	0.537 ± 0.091	0.537 ± 0.074	0.501 ± 0.059	0.491 ± 0.081
No. of female F1 weanlings examined	19	22	19	16
Final body weight (g)	60.59 ± 4.94	59.70 ± 4.16	59.11 ± 4.67	56.38 ± 4.66*
Brain (g)	1.454 ± 0.070	1.449 ± 0.062	1.459 ± 0.062	1.465 ± 0.058
	2.408 ± 0.133	2.435 ± 0.136	2.481 ± 0.201	2.626 ± 0.191**
Thymus (mg)	270.81 ± 41.17	257.86 ± 36.53	267.59 ± 51.30	237.79 ± 43.97
	446.96 ± 60.01	432.05 ± 52.78	451.49 ± 69.38	422.93 ± 56.57
Spleen (g)	0.314 ± 0.061	0.324 ± 0.062	0.313 ± 0.066	0.281 ± 0.043
	0.517 ± 0.084	0.544 ± 0.095	0.527 ± 0.087	0.502 ± 0.072

a: Mean ± SD.

b: Absolute organ weight.

c: Relative organ weight.

*: Significantly different from the control value, *, p<0.05; **, p<0.01.

Applicant's summary and conclusion

Conclusions:

The NOAEL for fertility 240 ppm (16-21 mg/kg bw/d) based on decreased sperm head counts and focal seminiferous tubule atrophy in F1 males at 1200 ppm (80-107 mg/kg bw/d). There were no effects on reproductive endpoints such as fertility, mating, gestation and birth index.

Executive summary:

The reproductive toxicity of dicyclohexyl phthalate (DCHP) was evaluated in a two generation test in which male and female Sprague-Dawley (SD) rats of parental (F0) and F1 generation were exposed to DCHP in the diet at concentrations of 0 (control), 240, 1200 or 6000 ppm. With regard to the effects on the F0 and F1 parental animals, changes included inhibition of body weight gain and food consumption, diffuse hypertrophy of hepatocytes, and hypertrophy of thyroidal follicular epithelial cells at the doses of 1200 ppm and 6000 ppm. The following changes were observed in the 6000 ppm group: increase weights of the liver and thyroid, increased hyaline droplets in the renal proximal tubular epithelium (F0 and F1 males), reduction of prostatic weight (F1 males), and diffuse and/or focal atrophy of testicular seminiferous tubules (F1 males). In addition, slight prolongation of the estrous cycle was noted in the F0 females of the 6000 ppm group, along with reduced spermatid head counts in the testes (homogenation resistant spermatids) in F1 male receiving doses of 1200 ppm or 6000 ppm. It is thought that the prolonged estrous cycle was secondary to the suppression of body weight gain. There were no test substance related changes in clinical signs and reproductive capability (mating, fertility, gestation and birth index), or in data for the delivery and lactational periods, or serum hormone levels. With regard to effects on the offspring, inhibition of body weight gain was found in the F1 and F2 6000 ppm, and decrease of anogenital distance (AGD) and appearance of areola mammae were observed in the F1 male 6000 ppm and F2 male receiving doses of 1200 ppm or 6000 ppm. No effects of DCHP treatment on the offspring were observed on results of clinical signs, the number of the pups delivered, sex ratio, viability, physical development, reflex and response tests, external abnormalities, organ weights, or necropsy findings. From the present study of DCHP administered to rats over two-generations, the no observed effect level (NOEL) for effects on the parental animals including the endocrine system, is considered to be 240 ppm. With regard to the reproductive toxicological effects on the parental animals, the NOEL is 240 ppm for males and 1200 ppm for females. For offspring, the NOEL values are concluded to be 240 ppm for males and 1200 ppm for females.

In coclusion, The NOAEL for fertility was 240 ppm (16 - 21 mg/kg bw/d; m-f) based on decreased sperm head counts and focal seminiferous tubule atrophy in F1 males at 1200 ppm (80-107 mg/kg bw/d; m-f). The maternal NOAEL was 240 ppm based on changes in body and liver weights at 1200 ppm. For developmental effects, the NOAEL was 240 ppm (16-21 mg/kg bw/d; m-f), and LOAEL was 1200 ppm (80-107 mg/kg bw/d; m-f) based on decreased anogenital distance and retained nipples in F2 males.