Diundecyl phthalate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.35 (Two-Generation Reproduction Toxicity Test)

Version / remarks:

EC Dangerous Substances Directive (67/548/EEC), Annex V, Part B

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich

EC Number:

271-091-4

EC Name:

1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich

Cas Number:

68515-49-1

IUPAC Name:

bis(8-methylnonyl) phthalate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD BR - VAF/PLUS®

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Stone Ridge, New York (males) and Portage, Michigan (females). In study B male rats were purchased from the Raleigh Facility, Raleigh, North Carolina.
- Age at study initiation: Not reported
- Weight at study initiation: not reported
- Housing: Animals were individually housed in suspended stainless steel and wire mesh cages.
- Diet (e.g. ad libitum): certified rodent chow (PMI Feeds, Inc., Richmond, IN), ad libitum.
- Water (e.g. ad libitum): automatic watering system, ad libitum.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25ºC
- Humidity (%): 40-70 %
- Photoperiod: 12-h light/dark cycle

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly (stability data confirmed that DIDP was stable in feed at room temperature for at least 14 days)
- Mixing appropriate amounts with (Type of food): certified rodent chow (PMI Feeds, Inc., Richmond, IN).
- Storage temperature of food: Not reported

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: The mating period ended when all females were confirmed mated or approx. 2 weeks had elapsed.
- Proof of pregnancy: copulatory plug or sperm in a vaginal rinse referred to as gestational day (GD) 0.
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): After confirmation of mating, each mated female was returned to its own cage.
- Any other deviations from standard protocol: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test material was verified by gas chromatography and infrared analysis. Stability data confirmed that DIDP was stable in feed at room temperature for at least 14 days.
The actual concentrations used in the first study, based on dietary analysis, were 0.200 ± 0.010%, 0.401 ± 0.018%, and 0.807 ± 0.037%. In the second study the analytically confirmed concentrations were 0.019 ± 0.001%, 0.058 ± 0.002%, 0.192 ± 0.010% and 0.387 ± 0.019%.

Duration of treatment / exposure:

Males P1 and P2: 10 weeks prior to mating, through mating and until the day prior to euthanasia, following delivery of the last litter they sired.
Females P1 and P2: 10 weeks prior to mating, through mating, gestation and lactation and until sacrifice after weaning of offspring animals on PND 21.

Frequency of treatment:

constant exposure 7 days/week

Details on study schedule:

- Selection of parents from F1 generation when pups were 21 days of age.
Each litter was represented by at least one pup per sex if possible. Sibling matings were avoided.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: %

Remarks:

nominal in diet, study A and B

Doses / concentrations 2

Dose / conc.:

0.02 other: %

Remarks:

Nominal in diet, study B

Doses / concentrations 3

Dose / conc.:

0.06 other: %

Remarks:

nominal in diet, study B

Doses / concentrations 4

Dose / conc.:

0.2 other: %

Remarks:

nominal in diet, study A and B

Doses / concentrations 5

Dose / conc.:

0.4 other: %

Remarks:

nominal in diet, study A and B

Doses / concentrations 6

Dose / conc.:

0.8 other: %

Remarks:

nominal in diet, study A

No. of animals per sex per dose:

P1 generation: 30
F1 generation: 30

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Dietary levels selected for the initial study A were based on results from a preliminary one-generation study. In this study, rats (10/sex/dose) were fed DIDP at dietary levels of 0.25, 0.50, 0.75, and 1.0% for approximately 10 weeks prior to mating and throughout the mating period. The males were sacrificed after mating, but females continued to be exposed throughout gestation and lactation, until weaning of the offspring on PND 21. Based on reductions in offspring and adult body weights at 0.75% and/or 1%, the dietary levels of DIDP selected for the initial two-generation study were 0.2, 0.4, and 0.8%.

Positive control:

N/A

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males: weekly. Females: weekly, during gestation on GD 0, 7, 14, and 21, and during lactation on PND 0, 4, 7, 14, and 21.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: weekly. Females: weekly, during gestation on GD 0, 7, 14, and 21, and during lactation on PND 0, 4, 7, 14, and 21..

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes. Parental food consumption was determined at the same time as the body weight measurements, except during mating.
- 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: No data

Oestrous cyclicity (parental animals):

The estrous cycle of each P1 and F1 female was evaluated daily by vaginal smears beginning three weeks prior to mating, continuing until the end of cohabitation, and on the day of sacrifice.

Sperm parameters (parental animals):

Parameters examined in P2 male generation:
Samples of sperm from the left distal caudal epididymis (or proximal vas deferens) were collected at scheduled terminal sacrifice from F2 male offspring and evaluated for the percentage of progressively motile sperm and sperm morphology. Also the entire left caudal epididymis was minced in saline to enumerate the total number of sperm (cauda reserves), and the left testis was homogenized to quantify the total number of homogenization resistant spermatids. Total cauda sperm counts and sperm motility were evaluated using the Hamilton-Thorne IVOS 2000 system for computer assisted sperm analysis (CASA).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4/sex/litter (as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
Litters from the P1 and F1 generations were examined twice daily for general appearance of the pups and dead offspring. All pups were counted, sexed, weighed, and examined externally on PND 0, 1, 4, 7, 14, and 21. Beginning on PND 29, all surviving F1 female offspring were examined daily for vaginal opening. These examinations continued until all animals reached criteria (i.e. vaginal opening).
In the Study B nipple retention and anogenital distance were measured, as were vaginal patency and preputial separation

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following delivery of the last litter they sired.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

GROSS NECROPSY
- Complete gross postmortem examinations were conducted on all animals in the study.

HISTOPATHOLOGY / ORGAN WEIGHTS
Liver, kidneys (paired), testes (individual), prostate, seminal vesicles,right epididymis (total and cauda), ovaries (individual), uterus, and brain from all parental animals that survived to scheduled termination were removed and weighed. The pituitary, testes, epididymides, prostate, seminal vesicles, vagina, uterus, ovaries, mammary gland, oviducts, thymus, adrenals, coagulating gland, kidney, liver and gross lesions from all parental animals in the control and 0.8% dose groups were examined microscopically.
In addition, the reproductive organs of all animals in the 0.2% and 0.4% dose groups that had abnormal sperm, estrous cycles, or failed to produce viable litters were examined. The testes of the P1 and F1 males were preserved in Bouin's solution. All other tissues were fixed in 10% neutral formalin. The tissues were processed, embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin. Five sections of each ovary from females in the control and high dose were examined for oocyte evaluation.
In the Study B pathologic examinations were limited to the organs with microscopic findings in the first study (i.e. liver and kidney).

Postmortem examinations (offspring):

SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
-Complete gross postmortem examinations were conducted on all animals in the study.

Statistics:

Quantitative continuous variables were analyzed for statistical significance. Bartlett's test of homogeneity of variance was used to determine if the groups had equal variance at the 1% level of significance. If the variances were equivalent, the groups were compared by using a standard one-way analysis of variance (ANOVA). If significant differences among the means were indicated, Dunnet's test was performed to determine which treated groups differed from control. In addition to the ANOVA, a standard regression analysis for linear response in the dose groups was performed. If the groups did not have equivalent variances at the 1% level, then a Kruskal-Wallis test (nonparametric) was used to assess differences in group means. If the means were different, Dunn's Rank Sum test (nonparametric) was used to determine which treatment groups differed significantly from control. In addition to the KW test, Jonckheere's test (nonparametric) for ordered response was also performed. Pup weight was analyzed by a mixed model of covariance with pups nested within dams, dams nested within dose, and total litter size as the covariant. If b.w. differences in groups were identified, the least squares means and the least significant difference test were used to determine which groups differed from the control group. Parental reproductive and offspring survival incidence data were evaluated by chi-square test analysis. Each treatment group was compared to the controls by using a 2x2 Fisher Exact test. Armitage's test for linear trend in the dosage groups was also performed.Sperm and oocyte count data were transformed by Blom’s transformation and analyzed by standard one way ANOVA. Residuals from the model were tested for normality by the Shapiro-Wilk test. Developmental landmarks were analyzed separately for each sex using a cumulative logistic regression model. Offspring survival was further assessed by fitting a modified probit model to the F2 offspring PND 0–4 survival data.

Reproductive indices:

Male mating index: no. males confirmed mated/no. males used for mating.
Male fertility index: no. males impregnating females/no. males used for mating.
Female fertility index: no. females confirmed mated/no. females paired.
Female fecundity index: no. females pregnant (excluding unconfirmed mated females)/no. females confirmed mated.
Gestational index: no. females with live litters/no. females pregnant.

Offspring viability indices:

Live birth index: no. live pups at birth/no. of pups born.
Day 1 survival index: no. live pups at Day 1/no. live pups at Day 0.
Day 4 survival index: no. live pups at Day 4/no. live pups at Day 0.
Day 7 survival index: no. live pups at Day 7/no. live pups at Day 4 (postcull).
Day 14 survival index: no. live pups at Day 14/no. live pups at Day 7.
Day 21 survival index: no. live pups at Day 21/no. live pups at Day 14.
Viability at weaning index: no. live pups at Day 21/no. live pups at Day 4 (postcull).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Study A and B: There were no important or dose dependent clinical signs of toxicity in either the P1 males or females in any dose group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Study A: All but two parental (P1) animals survived to scheduled termination. Although the causes of death were not determined, both were from the control group so clearly did not die as a consequence of treatment.
Study B: there were no treatment-related effects on parental survival.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Study A: Male and female body weights in the 0.8% dose groups were significantly (ca. 9 to 10%) below control values during the premating period, as were female body weights during gestation and lactation.
Study B: there were no differences in body weights.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Study A and B: there were no differences in food consumption.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Study A: Microscopic changes noted in male and female livers consisted of either centrilobular or diffuse hepatocellular hypertrophy, consistent with peroxisomal proliferation. There were also dose related increases in incidence and severity of histologic changes in male rat kidneys. These changes included accumulation of dark orange or eosinophilic granular cytoplasmic pigment in cortical tubules, cortical tubular degeneration, and increased incidence of granular casts in renal tubules in high dose males. These findings were consistent with alpha 2 u-globulin nephropathy. No histopathologic changes were observed in females. The only other microscopic change observed was thymic atrophy in females from the 0.8% group, judged to have been a secondary, stress related phenomenon.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Study A: There were no differences observed in mating, male or female fertility, gestational index, or length of gestation. Further, there were no differences in mean litter size or sex ratio.
Study B: There were no differences in the various reproductive indices, live birth indices, or male/female sex ratios.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

0.8 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: No adverse effects observed at the highest dose tested (equivalent to ca. 600 mg/kg bw/day)

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no important or treatment-related clinical findings.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Study A: All but two of the animals selected as parents for the second generation (a control male and a 0.4% group female) survived to scheduled termination.
Study B: There were no treatment-related deaths with the test material in any F1 animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Study A: Body weights were significantly reduced in comparison to controls among males at the 0.8% dose level throughout the study and at the 0.4% dose level at later time points (6 to14% decrease). Among the females, body weights were generally below control values (6% reduction) in the 0.8% dose group, but were only significantly different at postpartum days 10 and 14.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Study A: Adult organ weight data were similar to those noted in the P1 generation. There were significant increases in female liver weights in all dose groups and in high dose group males. Kidney weights were significantly elevated in males from all dose groups. Left ovary weights were reduced in females in the 0.8% dose group; however, the uterus weights were not significantly different when expressed as a fraction of body weight. Weights of male reproductive organs were not different from control when expressed on an absolute basis, but testes, epididymis, and seminal vesicle weights were above control values when expressed on a relative basis.
Study B: There was evidence of liver and kidney enlargement in both males and females at the 0.2% and 0.4% levels, similar to observations in the previous two-generation Study A

Gross pathological findings:

no effects observed

Description (incidence and severity):

Study A: There were no pathologic changes in sexual organs from either males or females.
Study B: There were no gross postmortem observations judged to have been related directly to treatment with the test material in any F1 animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Similar to the P1 animals, histopathologic alterations were noted in kidneys of treated males and in livers of treated animals of both sexes, but there were no histologic changes in other organs.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Study A: Length of estrus cycle and number of oocytes were unaffected by treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Study A: sperm count and sperm quality indices were unaffected by treatment.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Study A: Mating indices, fertility indices, gestational index, mean length of gestation, and mean litter size were unaffected by treatment. The sex ratio and live birth index were significantly decreased in the 0.2% dose group, but were unaffected in the 0.4% and 0.8% dose groups, suggesting that the findings in the 0.2% dose group were due to chance rather than treatment.
Study B: There were no significant differences in fertility indices, litter size, or sex ratio. Unlike the F2 offspring in Study A, no changes were observed in percent of live births at any dose level.

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Effect level:

0.8 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: No adverse effects observed at the highest dose tested (equivalent to ca. 600 mg/kg bw/day)

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity was noted in the offspring.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Study A: Offspring survival at PND 4 was significantly reduced in the 0.8% dose group (below the HCR) but was not reduced at later time points. Survival in other dose groups was generally better than the corresponding control values.
Study B: There were no differences in survival or viability at weaning during the postnatal period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Study A: Offspring body weights were also reduced in the 0.8% dose group at PND 0 and these values were statistically significant and outside the laboratory HCR. Body weights during the postnatal period were significantly below control values in the 0.8% group, but were generally within the HCR. By PND 35, the offspring body weights were similar to control values.
Study B: there were no differences in offspring body weights during the postnatal period to PND 35.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Study A: There was a statistically significant increase in age at vaginal patency in the 0.4 and 0.8% dose groups, but overall the difference was small (≤ 2 days) and within a range of unknown biologic relevance.
Study B: There were no differences in F1 offspring developmental landmarks, anogenital distance, nipple retention, preputial separation, or vaginal patency.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Study A: Organ weights were taken from randomly selected offspring at the end of weaning. Many of the organ weights were significantly below control values in the 0.8% group when expressed on an absolute basis; on a relative basis the brain and liver weights were above control values but there were no differences in any of the sexual organs.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Study A: No gross postmortem observations were noted in the offspring.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Study A: Dose related enlargement of hepatocytes with cytoplasmic eosinophilia was seen in the livers of 0.4% and 0.8% dose group offspring of both sexes. No treatment related microscopic findings were noted in kidneys or other organs.

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.8 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental landmarks

Remarks on result:

other: No adverse effects observed at the highest dose tested (equivalent to ca. 600 mg/kg bw/day)

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Study A: F2 offspring survival at PND 1 and 4 was significantly reduced in all treatment groups and continued to be reduced in the 0.8% dose group throughout the postnatal period. These decreases in offspring survival were generally outside the laboratory HCR.
Study B: As in the first study, F2 offspring survival was significantly reduced on PND 1 and 4 in the 0.2% and 0.4% dose groups and was below laboratory HCR. Offspring survival was generally unaffected over the remainder of the postnatal period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Study A: PND 0 body weights from male F2 offspring were reduced in the 0.8% dose group. Weight gain was significantly reduced in both male and female offspring in the 0.8% dose groups for the entire postnatal period. These decreases in PND 0 body weights and F2 offspring body weights were generally below the laboratory HCR.
Study B: all treated males and females from the 0.2% and 0.4% dose groups had mean body weights that were outside the laboratory HCR on PND 0. However, none of the values was statistically significantly different from concurrent controls, and there were no consistent dose-response relationships. There were some significant reductions in F2 offspring body weights at PND 14 and 21 in the 0.2 and 0.4% dose groups (6 to 9% difference), but these values were within the HCR and were transient. There were no significant differences in 0.4% dose group female body weights at PND 28 or in male body weights at PND 35 and 42. Overall, the effect on offspring body weights was judged to have been treatment-related due to the dose-response relationship and the similarity to decreases observed in the one-generation study in the 0.5% dose group. However, because the differences were within the HCR and transient, they were not considered to be toxicologically important.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

Study B: There were no differences in anogenital distance, nipple retention, or vaginal patency in the F2 offspring. Preputial separation was slightly delayed in the 0.4% dose group (1.2 days). Although this difference was statistically significant, it was deemed not adverse because the magnitude was so small.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Study A: Histopathologic changes in livers were noted in 0.4% and 0.8% dose groups of F2 offspring, similar to those previously seen in F1 offspring.

Other effects:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

0.06 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Remarks on result:

other: The NOAEL is equivalent to ca. 50 mg/kg bw/day.

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1. Selected Organ Weights of Adult Male and Female Rats from the Two-Generation Reproductive Study A (g)

	MALES—P1 GENERATION
Dose (%)	Liver	Kidneys	Prostate	Left Testis	Right Testis	Right Total Epididymis	Right Cauda Epididymis	Seminal Vesicles
0	23.48±3.18	4.62±0.40	0.816±0.145	1.87±0.18	1.90±0.17	0.8276±0.0823	0.329±0.049	2.89±0.50
0.2	24.11±3.23	5.03±0.47**	0.821±0.186	1.88±0.14	1.90±0.16	0.8314±0.0839	0.349±0.052	3.04±0.34
0.4	25.96±4.07*	5.51±0.60**	0.796±0.143	1.87±0.20	1.87±0.20	0.8469±0.1015	0.375±0.034**	2.94±0.44
0.8	27.91±3.21**	5.71±0.52**	0.773±0.174	1.93±0.15	1.94±0.16	0.8635±0.0719	0.380±0.043**	2.85±0.44
	MALES—P2 GENERATION
Dose (%)	Liver	Kidneys	Prostate	Left Testis	Right Testis	Right Total Epididymis	Right Cauda Epididymis	Seminal Vesicles
0	23.50±3.00	4.43±0.38	0.84±0.17	2.06±0.13	2.08±0.13	0.7898±0.0708	0.223±0.046	2.85±0.41
0.2	23.81±3.53	4.82±0.55*	0.83±0.23	2.01±0.30	2.06±0.19	0.8762±0.1273	0.222±0.049	2.87±0.48
0.4	25.22±4.03	5.24±0.65**	0.86±0.19	2.00±0.21	2.02±0.22	0.8927±0.0822	0.236±0.032	3.04±0.41
0.8	26.36±3.09*	5.29±0.66**	0.80±0.14	1.99±0.17	2.00±0.18	0.9122±0.0842	0.237±0.040	2.98±0.55

	FEMALES—P1 GENERATION
Dose (%)	Liver	Kidneys	Uterus	Right Ovary	Left Ovary
0	17.53±1.74	3.04±0.21	0.69±0.18	0.0557±0.0104	0.0636±0.0115
0.2	19.07±1.68*	3.13±0.26	0.62±0.16	0.0618±0.0131	0.0588±0.0132
0.4	20.64±1.45**	3.27±0.27*	0.66±0.20	0.0634±0.0121	0.0581±0.0102
0.8	21.00±2.02**	3.08±0.36	0.53±0.21*	0.0481±0.0118	0.0471±0.0127**
	FEMALES—P2 GENERATION
Dose (%)	Liver	Kidneys	Uterus	Right Ovary	Left Ovary
0	21.00±1.55	3.27±0.29	0.53±0.08	0.0689±0.0122	0.0658±0.0135
0.2	24.66±2.62**	3.67±0.48**	0.64±0.13	0.0711±0.0156	0.0688±0.0113
0.4	24.82±2.99**	3.78±0.34**	0.69±0.17**	0.0699±0.0135	0.0671±0.0138
0.8	25.54±2.35**	3.54±0.35	0.49±0.15	0.0583±0.144*	0.0532±0.0165*

Values are means ± SD

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P < 0.01)

Table 2. Results of Reproductive Indices, Live Birth Index, and Postnatal Survival Indices of the P1 Generation from the Two-Generation Reproductive Study A (g)

Dose (%)	Male Mating (%)	Male Fertility (%)	Female Mating (%)	Female Fertility (%)	Gestational Index (%)	Mean Gestation (Days)	Mean Litter Size	Mean Live Offspring	Mean Dead Offspring	Live males (%)	Live Females (%)
0	88.0	76.0	88.0	81.8	89.5	22.3	15.3	15.1	0.2	48.8	51.2
0.2	93.3	76.7	93.3	78.6	100.0	22.4	14.7	14.3	0.3	54.8	45.2
0.4	80.0	76.7	80.0	91.7	100.0	22.3	14.7	14.3	0.4	48.2	51.8
0.8	88.0	86.0	88.0	90.9	100.0*	22.2	14.9	14.0	0.9	48.3	51.7

F1 OFFSPRING
Dose (%)	Live Birth (%)	Day 1 Survival (%)	Day 4 Survival (%)	Day 7 Survival (%)	Day 14 Survival (%)	Day 21 Survival (%)	Viability at Weaning (%)
0	98.7	95.5	93.9	97.8	95.5	100.0	93.4
0.2	97.6	95.8	93.0	100.0	100.0**	100.0	100.0**
0.4	96.8	94.2	91.5	99.4	99.4*	100.0	98.9*
0.8	94.2**	92.2	88.8*	98.0	98.4	100.0	96.4
HCR	95.2–99.2	96.2–100	92.8–99.7	97.8–100	93.7–100	98.8–100	86.9–100

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P< 0.01)

KEY:

Male Mating Index: # males confirmed mated/#males used for mating

Male Fertility Index: # males impregnating females/# males used for mating

Female Mating Index: # females confirmed mated/# females paired

Female Fertility Index: # females pregnant (excluding unconfirmed mated females)/# females confirmed mated

Gestational Index: # females with live litters/# females pregnant

Live Birth Index: # live pups at birth/# of pups born

Day 1 Survival Index: # live pups at Day 1/# live pups at Day 0

Day 4 Survival Index: # live pups at Day 4/# live pups at Day 1

Day 7 Survival Index: # live pups at Day 7/# live pups at Day 4 (postcull)

Day 14 Survival Index: # live pups at Day 14/# live pups at Day 7

Day 21 Survival Index: # live pups at Day 21/# live pups at Day 14

Viability at Weaning Index: # live pups at Day 21/# live pups at Day 4 (post cull)

Table 3A. Time to Vaginal Patency During the Weaning Phase of the F1 Generation from the Two-Generation Reproductive Study A (days)

Dose (%)	Age at Vaginal Patency
0	32.2±1.4
0.2	32.4±2.1
0.4	33.5±2.8*
0.8	34.2±2.3**

Values are means ± SD

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P < 0.01)

Table 3B. Summary of Sperm Count, Sperm Quality Indices, Estrus Cycle Length, and Oocyte Count of the P2 Generation from the Two-Generation Reproductive Study A

Dose (%)	Resistant Spermatid Count (106/gram of testes)	TCSC (x 108)	Percent Sperm Motility	Percent Normal Sperm Morphology	Estrous Cycle Length (Days)	Normal Cycles (%)	Oocyte Counts
0	135.2	8.57	60.2	93.2	4.18	96.7	114.4
0.2	131.7	7.94	58.2	92.2	4.13	90.0	NE
0.4	129.9	8.43	60.8	95.7	4.17	90.0	NE
0.8	124.5	8.43	60.8	95.9	4.31	100.0	135.8

TCSC = Total Cauda Sperm count

NE = Not Examined

Table 4. Results of Reproductive Indices, Live Birth Index, and Postnatal Survival Indices of the P2 Generation from the Two-Generation Reproductive Study A

Dose (%)	Male Mating (%)	Male Fertility (%)	Female Mating (%)	Female Fertility (%)	Gestational Index (%)	Mean Gestation (Days)	Mean Litter Size	Mean Live Offspring	Mean Dead Offspring	Live males (%)	Live Females (%)
0	82.8	65.5	80.0	75.0	100.0	22.4	14.3	14.1	0.2	54.3	45.7
0.2	90.0	70.0	90.0	77.8	100.0	22.4	15.2	14.4	0.8	41.7**	58.3**
0.4	83.3	66.7	83.3	76.0	100.0	22.4	14.5	14.2	0.3	51.1	48.9
0.8	100.0*	93.3*	100.0*	93.3	100.0	22.1	14.5	14.0	0.5	55.6	44.4

F2 OFFSPRING
Dose (%)	Live Birth (%)	Day 1 Survival (%)	Day 4 Survival (%)	Day 7 Survival (%)	Day 14 Survival (%)	Day 21 Survival (%)	Viability at Weaning (%)
0	98.5	96.6	94.0	99.3	99.3	100.0	98.7
0.2	94.7*	92.1*	85.8**	100.0	100.0	100.0	100.0
0.4	98.2	89.6**	86.7**	99.3	98.5*	100.0	97.8
0.8	96.8**	85.2**	77.6**	95.4*	98.4	98.9	92.9*
HCR	95.2–99.2	96.2–100	92.8–99.7	97.8–100	93.7–100	98.8–100	86.9–100

Key same as Table 2

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P < 0.01)

Table 5. Selected Organ Weights of Adults and Offspring from the Two-Generation Reproductive Study B (g)

ADULTS
	P1 Generation				P2 Generation
	Male		Female		Male		Female
Dose (%)	Liver	Kidneys	Liver	Kidneys	Liver	Kidneys	Liver	Kidneys
0	21.27±3.04	4.14±0.36	17.57±2.54	3.07±0.31	23.61±4.06	4.64±0.6	15.93±1.60	2.98±0.26
0.02	21.03±2.59	4.06±0.38	16.95±2.37	2.97±0.19	23.22±4.22	4.68±0.47	15.85±2.02	3.01±0.26
0.06	21.31±2.57	4.12±0.35	17.41±3.48	2.98±0.26	23.42±4.01	4.64±0.58	16.12±1.61	3.07±0.30
0.2	22.26±2.68	4.36±0.43	18.21±2.68	3.07±0.30	25.15±3.48	5.09±0.54*	18.68±2.28**	3.38±0.44**
0.4	23.90±2.70**	4.74±0.45**	19.61±2.71*	3.24±0.28	26.60±3.98*	5.56±0.53*	19.54±2.47**	3.15±0.45
OFFSPRING
	P1 Generation				P2 Generation
	Male		Female		Male		Female
Dose (%)	Liver	Kidneys	Liver	Kidneys	Liver	Kidneys	Liver	Kidneys
0	2.38±0.51	0.72±0.10	2.18±0.35	0.70±0.08	2.00±0.47	0.65±0.11	1.90±0.35	0.65±0.11
0.02	2.35±0.41	0.74±0.09	2.14±0.27	0.71±0.07	2.15±0.42	0.71±0.13	2.10±0.49	0.70±0.13
0.06	2.26±0.47	0.70±0.13	2.00±0.47	0.68±0.14	2.11±0.41	0.67±0.10	1.89±0.38	0.63±0.10
0.2	2.39±0.40	0.72±0.10	2.24±0.42	0.73±0.12	2.08±0.44	0.66±0.11	1.95±0.42	0.62±0.10
0.4	2.32±0.58	0.72±0.13	2.17±0.47	0.68±0.11	1.93±0.54	0.63±0.12	2.05±0.54	0.63±0.11

Values are means ± SD

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P < 0.01)

Table 6. Results of Reproductive Indices, Live Birth Index, and Postnatal Survival Indices of the P1 Generation from the Two-Generation Reproductive Study B

Dose (%)	Male Mating (%)	Male Fertility (%)	Female Mating (%)	Female Fertility (%)	Gestational Index (%)	Mean Gestation (Days)	Mean Litter Size	Mean Live Offspring	Mean Dead Offspring	Live males (%)	Live Females (%)
0	90.0	86.7	90.0	92.6	10.0	22.6	14.7	14.2	0.5	50.3	49.7
0.02	90.0	96.7	90.0	100.0	96.6	22.5	14.2	14.0	0.2	46.2	53.8
0.06	79.3	89.7	79.3	100.0	92.3	22.4	15.5	15.4	0.1	50.0	50.0
0.2	96.7	86.7	96.7	89.7	100.0	22.4	14.7	14.0	0.6	49.9	50.1
0.4	83.3	80.0	83.3	84.0	100.0	22.3	14.8	14.6	0.1	52.4	47.6

F1 OFFSPRING
Dose (%)	Live Birth (%)	Day 1 Survival (%)	Day 4 Survival (%)	Day 7 Survival (%)	Day 14 Survival (%)	Day 21 Survival (%)	Viability at Weaning (%)
0	96.3	97.8	96.2	99.5	100.0	100.0	99.5
0.02	98.5	98.5	97.8	100.0	99.5	99.5	99.0
0.06	99.2*	98.4	95.9	99.5	100.0	100.0	99.5
0.2	95.8	95.9	95.3	100.0	100.0	100.0	100.0
0.4	99.2*	97.7	96.9	99.5	100.0	100.0	99.5
HCR	95.2–99.2	95.5–100	88.9–99.5	92.8–100	93.7–100	98.8–100	86.9–100

*Mean significantly different from controls (P < 0.05)

Table 7. Time to Offspring Developmental Landmarks from the Two-Generation Reproductive Study B

F1 OFFSPRING
Dose (%)	Anogential distance at PND 0 (mm)		Nipple retention at PND 12–13 (Number of thoracic nipples)		Age at Preputial Separation (Days)	Age at Vaginal patency (Days)
	Males	Females	Males	Females
0	1.98±0.39	0.90±0.17	0±0.00	6.01±0.10	43.4±2.3	31.9±0.8
0.02	2.02±0.36	0.92±0.17	0±0.00	6.00±0.00	43.0±1.7	31.7±1.3
0.06	1.98±0.35	0.91±0.15	0±0.00	6.00±0.00	43.0±2.0	31.5±1.2
0.2	2.05±0.34	0.93±0.16	0±0.00	5.97±0.30	43.3±1.7	31.9±1.6
0.4	2.00±0.35	0.94±0.17	0±0.00	6.00±0.00	43.7±2.2	31.9±1.2
F2 OFFSPRING
Dose (%)	Anogential distance at PND 0 (mm)		Nipple retention at PND 12–13 (Number of thoracic nipples)		Age at Preputial Separation (Days)	Age at Vaginal patency (Days)
	Males	Females	Males	Females
0	2.30±0.30	1.05±0.16	0±0.00	6.01±0.10	44.7±2.8	32.1±1.1
0.02	2.31±0.26	1.01±0.15	0±0.00	6.00±0.00	44.0±1.6	31.9±1.1
0.06	2.23±0.29	1.02±0.14	0±0.00	6.00±0.00	44.6±1.9	31.6±0.9
0.2	2.33±0.30	1.05±0.15	0±0.00	6.00±0.00	45.2±2.0	32.1±1.2
0.4	2.31±0.31	1.06±0.16	0±0.00	6.03±0.23	45.9±2.6*	32.2±1.4

Values are means ± SD

*Mean significantly different from controls (P < 0.05)

Table 8. Results of Reproductive Indices, Live Birth Index, and Postnatal Survival Indices of the P2 Generation from the Two-Generation Reproductive Study B

Dose (%)	Male Mating (%)	Male Fertility (%)	Female Mating (%)	Female Fertility (%)	Gestational Index (%)	Mean Gestation (Days)	Mean Litter Size	Mean Live Offspring	Mean Dead Offspring	Live males (%)	Live Females (%)
0	90.0	83.3	90.0	88.9	100.0	22.3	15.9	15.6	0.4	50.4	19.6
0.02	90.0	86.7	90.0	96.3	96.2	22.3	15.2	15.0	0.2	52.3	47.7
0.06	93.3	83.3	93.3	89.3	100.0	22.4	15.6	15.2	0.4	51.8	48.2
0.2	86.7	70.0	86.7	76.9	100.0	22.3	14.9	14.8	0.1	53.9	46.1
0.4	96.7	90.0	96.7	93.1	100.0	22.1	15.9	14.9	0.7	53.0	47.0

F2 OFFSPRING
Dose (%)	Live Birth (%)	Day 1 Survival (%)	Day 4 Survival (%)	Day 7 Survival (%)	Day 14 Survival (%)	Day 21 Survival (%)	Viability at Weaning (%)
0	97.7	99.0	97.7	98.5	95.4	100.0	94.0
0.02	98.7	98.4	96.8	99.0	99.5*	100.0	98.5
0.06	97.4	97.4	96.6	99.0	100.0*	99.5	98.5*
0.2	99.4	95.2**	92.3**	98.8	98.8	98.7	96.3
0.4	95.5	89.1**	84.8**	99.0	98.5	98.5	96.0
HCR	95.2–99.2	95.5–100	88.9–99.5	92.8–100	93.7–100	98.8–100	86.9–100

Key same as Table 2

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P < 0.01)

Applicant's summary and conclusion

Conclusions:

In 2 two-generation reproduction toxicity studies, di-isodecyl phthalate (DIDP) was determined to have a NOAEL of 0.8% (approximately 600 mg/kg/day) for fertility, reproduction function and developmental landmarks and 0.06% (approximately 50 mg/kg/day) for F2 offspring survival.

Executive summary:

Reproductive toxicity of di-isodecyl phthalate (DIDP) was evaluated in 2 two-generation reproductive toxicity studies in accordance with EU Method B.35 and EPA OPPTS 870.3800 and GLP standards. In the first study, dietary levels of 0.2%, 0.4% and 0.8% DIDP were administered in 30 Sprague-Dawley rats per sex and per dose of generation P1 and generation P2. A second study with lower dietary levels of 0.02, 0.06, 0.2, and 0.4% DIDP was performed similarly to the first study. In both studies, there were no changes in reproductive indices and no effects on fertility in the P1 or P2 generation. There were decreases on adult body weight, but no consistent clinical signs and no mortality. In both studies, liver and kidney effects were observed in the P1 generation. Increased liver weights and associated hepatocellular hypertrophy were observed at dietary concentrations of 0.4% and greater in both studies. These dietary concentrations also produced kidney effects that were associated with alpha 2u-globulin toxicity, a male rat specific effect and thus not relevant to humans. There were no effects on live birth index, but reduced offspring survival was observed at postnatal days 1 to 4. This reduced survival was more pronounced in the F2 generation in which statistical significance was achieved at levels of 0.2% DIDP and greater. Because of these results observed in the first study, as well as the apparent lack of correlation with other treatment-related effects, a second study was carried out but the same results were obtained in F2 survival. There were also transient decreases in offspring body weights prior to weaning, corresponding to rapid offspring growth, and high levels of food consumption. There were no notable alterations in developmental landmarks. In conclusion, these studies provided a NOAEL of 0.06% (approximately 50 mg/kg/day) for F2 offspring survival and 0.8% (approximately 600 mg/kg/day) for fertility, other measures of reproductive function, and developmental landmarks.