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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A GLP compliant study undertaken to a standard considered to be equivalent to international test guidelines for a two-generation rat reproduction study.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2000
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: EC Dangerous Substances Directive (67/548/EEC), Annex V, Part B; 1987
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4700 (Reproduction and Fertility Effects)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich
EC Number:
271-090-9
EC Name:
1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich
Cas Number:
68515-48-0
Molecular formula:
C26 H42 O4
IUPAC Name:
Not available, not a single isomer
Constituent 2
Reference substance name:
DINP
IUPAC Name:
DINP
Details on test material:
- Name of test material (as cited in study report): Di-isononyl phthalate (DINP)
- Substance type: technical material
- Physical state: not reported
- Analytical purity: >99.7% verified by gas chromatography and infrared analysis
- Impurities (identity and concentrations): did not contain any contaminants that could affect the outcome of the study
- Composition of test material, percentage of components: N/A
- Purity test date: not stated
- Lot/batch No.: not stated
- Expiration date of the lot/batch: not stated
- Stability under test conditions: stability data confirmed that DINP was stable in feed for at least 14 days
- Storage condition of test material: not stated
- Other:

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Stone Ridge, NY, USA
- Age at study initiation: (P) x wks not reported; (F1) x wks not reported
- Weight at study initiation: (Means given; two gen study): (P) Males: 202.1 - 204.1g; Females: 158.4 - 159.6g; (F1) Males: 247.8 - 249.2g; Females: 178.0 - 180.3g; (F2) Males: 184.6 - 211.1g; Females: 150.3 - 167.5g
- Fasting period before study: N/A
- Housing: individually housed in suspended stainless steel and wire mesh cages prior to mating
- Diet (e.g. ad libitum): ad libitum (free access)
- Water (e.g. ad libitum): ad libitum (free access)
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 - 76°C
- Humidity (%): 40 - 70 %
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light


IN-LIFE DATES: Not reported

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): biweekly
- Mixing appropriate amounts with (Type of food): control (0.0%), 0.2, 0.4 or 0.8% in Certified Rodent Chow (PMI Feeds, Richmond, IN, USA)
- Storage temperature of food: not reported


VEHICLE N/A


Details on mating procedure:
- M / F ratio per cage: 1:1
- Length of cohabitation: up to evidence of mating by either observation, copulatory plug or sperm in a vaginal rinse or up to about 3 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged : not reported
- Any other deviations from standard protocol: N/A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dietary analysis over the course of the study indicated concentration values of DINP to be 0.193 ± 0.008%, 0.390 ± 0.011%, and 0.782 ± 0.038% in comparison to target levels of 0.2, 0.4 and 0.8%.
Duration of treatment / exposure:
Constant exposure up to about 10 weeks prior to mating, continuing throughout the mating period. Males were sacrificed after mating but treatment of the females continued throughout gestation and lactation until weaning of the offspring on postnatal day (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.
- Age at mating of the mated animals in the study: not reported
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.2
Basis:
nominal in diet
% DINP
Remarks:
Doses / Concentrations:
0.4
Basis:
nominal in diet
% DINP
Remarks:
Doses / Concentrations:
0.8
Basis:
nominal in diet
% DINP
No. of animals per sex per dose:
30 per sex per group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: results from a preliminary one generation study demonstrated increased adult liver and kidney weights at dietary levels 0.5%, and body weight gain was reduced at 1% and 1.5%. It was anticipated that the body weight gain of both adults and offspring would be significantly reduced at the 0.8% level.
- Rationale for animal assignment (if not random): randomly allocated
Positive control:
N/A

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week; during gestation females were examined on gestation day 0, 7, 14 and 21 and postnatal days 0, 4, 7, 14 and 21.


BODY WEIGHT: Yes
- Time schedule for examinations: once per week; during gestation and lactation female bodyweights were recorded on gestation day 0, 7, 14 and
21 and postnatal days 0, 4, 7, 14 and 21.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Parental food consumption was measured weekly except during gestation when
female food consumption was measured at three to four day intervals.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No



Oestrous cyclicity (parental animals):
N/A
Sperm parameters (parental animals):
N/A
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
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

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 it sired.
- Maternal animals: All surviving animals after the last litter of each generation was weaned


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera


HISTOPATHOLOGY / ORGAN WEIGHTS
The relevant tissues were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [?] days of age. Not reported
- These animals were subjected to postmortem examinations macroscopic and microscopic examination as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera and brain.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively.
Organ weights of the following in all animals: Liver, kidneys (paired), testes (individual), prostate, seminal vesicles, epididymides (individual), ovaries (individual), and brain.
Microscopic examinations of the following in the control and high level DINP (0.8%): Pituitary, testes, epididymides, prostate, seminal vesicles, vagina, uterus, ovaries, mammary gland and gross lesions.
Statistics:
Quantitative continuous variables (body weights, food consumption, organ weights, and relative organ weights) 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 Kruskal-Wallis 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. The mathematical model incorporates the litter size as an independent variable related to pup weight. If body weight 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. Pup weights were analyzed separately by sex.

Parental reproductive and offspring survival incidence data were evaluated by chi-square test analysis to determine if the proportions of incidences differed between the groups tested. 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. All tests were reported at the 5% or 1% level of significance.
Reproductive indices:
Mating, male and female fertility, fecundity, gestational index or length of gestation were not significantly different from control values in either generation of the two-generation reproductive toxicity study at the highest concentration of DINP tested (0.8% in the diet, ~ 500 mg/kg/day).
Offspring viability indices:
P1 effects: There was no apparent effect of treatment on survival. There were no important or dose-dependent clinical signs of toxicity in either P1 males or females or in any of their offspring.
F1 effects: Sex ratios and survival during lactation were unaffected by treatment. Mean litter sizes of the three treated groups were statistically significantly greater than the control group. Each treatment group contained 15 to 20% more pups than the controls between birth and culling (PND 4). At PND 0, weights were significantly reduced in male offspring from the 0.8% group but were within the historical control range.
Significant reductions in body weights were also noted in the 0.4% and 0.8% groups on PND 7 and 14 and in all three treatment groups at PND 21, but all of the weights were within the historical control range.
F2 effects: PND 0 offspring weights in the second generation were not significantly different from controls when litter size differences were taken into account, but were below the historical control range in the 0.8% group. Offspring body weights during lactation were significantly reduced in the 0.4% and 0.8% groups by comparison to control values. No gross effects related to treatment were recorded at necropsy.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no apparent effect of treatment on survival. All but two parental (P1) animals (one male from the 0.4% group and one female from the 0.2% group). There were no important or dose-dependent clinical signs of toxicity in either P1 males or females.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights were unaffected during premating and mating and, among the females, gestation. However, dams in the 0.8% group had significantly reduced body weights at postpartum days 14 and 21.
Although food consumption was recorded there is no data reporting any effects on this parameter.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No data

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no statistically significant differences in male mating, male fertility, female fertility, female fecundity or gestational indices.
Additionally, there were no differences among groups in the mean length of gestation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Statistically significant elevations were found in absolute liver weights: 0.8% DINP levels Males and 0.4 and 0.8% DINP levels Females.
Statistically significant elevations were found in absolute kidney weights: 0.4 and 0.8% DINP levels Males and 0.2, 0.4 and 0.8% DINP levels Females
Ovarian weight was reduced in females at the 0.8% DINP level. but the difference was only in the left ovary.
Relative weights were not statistically different. Absolute weights of testes, epididymides, prostate and seminal vesicles were unaffected.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No effects noted

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic examination of the livers revealed a minimal to moderately increased cytoplasmic eosinophilia in males and females from all DINP treated groups.

OTHER FINDINGS (PARENTAL ANIMALS)
None

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: body weight gain
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Fertility - No adverse effects observed.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)
No effects reported

CLINICAL SIGNS (OFFSPRING)
No effects reported

BODY WEIGHT (OFFSPRING)
The mean body weights of males in the 0.4% DINP group were 5-6% below control values but only achieved statistical significance sporadically. The mean female body weights in the same group were about 6% below control values but not significantly different from controls at any time after the first week. At the 0.8% level, the mean body weights of the males were significantly below control values throughout the pre-mating period, but the difference narrowed from ca. 13 to 7%. Among the females at the 0.8% level, the mean body weights were ca. 10% below and statistically different from control values at Week 0 and remained below control values during pre-mating, although the differences were not significantly different after the third week. This pattern continued through gestation. During lactation, the body weights of the dams were significantly below control values in the 0.8% group.

SEXUAL MATURATION (OFFSPRING)
There were no effects on any of the classic reproductive indices.

ORGAN WEIGHTS (OFFSPRING)
Increased mean kidney weights (statistically different from control means) were observed in males at the 0.8% DINP diet and statistically significant increased mean liver weights in females at the 0.8% level.

GROSS PATHOLOGY (OFFSPRING)
No gross effects were noted during autopsy

HISTOPATHOLOGY (OFFSPRING)
Examination of the kidneys revealed an increased incidence of minimal to moderate renal pelvis dilatation (primarily right kidney) in the males at 0.4% and 0.8% groups. This was not observed in females and is considered to be likely related to the induction of alpha 2u-globulin. No histological changes were seen in the testes or accessory organs or any of the other organs from either generation.

OTHER FINDINGS (OFFSPRING)
None reported

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 200 - <= 260 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: bodyweight gain
Dose descriptor:
BMC05
Generation:
F1
Effect level:
>= 200 - <= 260 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: The 95% lower confidence limit for a 5% reduction in the predicted body weights ranges from 0.16 to 0.21 % in the diet.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
This study showed that DINP treatment does not affect fertility or male reproductive development at doses of up to approximately 500 mg/kg body weight/day.
Executive summary:

The potential reproductive toxicity of di-isononyl phthalate (DINP) was assessed in a two-generation reproductive toxicity study. Groups of 30 male and female CRL : CD(SD)BR rats were given DINP via dietary administration at levels 0.0, 0.2, 0.4 or 0.8%. There were no changes in any of the classic reproductive parameters, i.e. mating, male or female fertility, fecundity, gestational index, or length of gestation. The overall NOAELs for these effects were the highest dietary level (%) tested, equivalent to approximately 500 mg/kg/day. There were no testicular effects in either the P1 males, exposed as juveniles and young adults or the P2 (F1) offspring exposed in utero, through lactation, and continuously to terminal sacrifice. Adult survival was unaffected at any level of dietary DINP. Liver and kidney weights were elevated at dietary level (%) above ca. 110 mg/kg/day, consistent with evidence from other studies of peroxisomal proliferation at these levels. The LOAEL of 159 mg/kg/day is conservative by nature since the dose of

interest actually consists of a range, 159 – 395 mg/kg/day, leading to the possibility that the NOAEL is on the border of the range or even within it. To address the issue of only having a LOAEL for decreases in pup body weight, the data were evaluated by benchmark dose procedures (Waterman et al., 2000). Benchmark dose modeling is a useful alternative since it makes no particular assumption about the nature of toxicological dose-responses, other than that the change in response generally does not decrease with higher doses. In particular, there is no specific assumption of the relationship between a putative no-effect level in the dose-response and the benchmark dose. The 95% lower confidence limit for a 5% reduction in the predicted body weights ranged from 0.16-0.21 % in

the diet (~200 – 260 mg/kg/day) and is appropriate for use in risk assessments. Therefore the value of 200 mg/kg/day will be used for risk characterisation.

In this study, neither fertility or male reproductive development was affected when DINP was administered to rats at a dietary dose level of up to approximately 500 mg/kg/day.