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

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is rated a "1" because it applied GLP, used appropriate testing procedures, and followed an accepted test guideline.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

Range finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Study Initiation: 8 June 1994
Inlife Test Period: 13 June 1994 to 12 October 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

P1 males and females received test material daily for at least ten weeks prior to mating and during the mating period. Additionally, P1 female animals received test material during the gestation and postpartum periods, until weaning of the F1 offspring on Postpartum Day (PPD) 21

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The rat is among the species of choice for reproduction and fertility testing according to the E.C Dangerous Substances Directive (67/548/EEC), Annex V part B "Two-Generation Reproduction Toxicity Test", and the U.S EPA test guidelines for reproduction and fertility effects (40 CFR, Part 798).

The dietary route is an accepted route of administration according to E.C. Dangerous Substances Directive Annex V, and U.S. EPA TSCA regulations, and represents a likely route of human exposure.

Specific details on test material used for the study:

Expiration Date: April 1999
Description: Colorless liquid
Storage Condition: Room temperature
The test material was assumed 100% pure for the purpose of dosing.

Species:

rat

Strain:

other: Crl:CD®BR-VAF/Plus

Details on species / strain selection:

The rat is among the species of choice for reproduction and fertility testing according to the E.C Dangerous Substances Directive (67/548/EEC), Annex V part B "Two-Generation Reproduction Toxicity Test", and the U.S EPA test guidelines for reproduction and fertility effects (40 CFR, Part 798).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories,Inc. Kingston facility, Stone Jdge, New York.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 7 wks
- Weight at study initiation: (P1) Males: 214.9 - 248.9 g; Females: 158.6 - 191.9 g
- Housing: Individually housed during the test period, except during the mating and postpartum periods.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Purina Certified Rodent Chow (5002 meal), ad libitum
- Water (e.g. ad libitum): Automatic watering system, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

other: Purina Certified Rodent Chow (5002 Meal)

Details on exposure:

Mixing of feed: The basal diet consisted of Certified Rodent Chow (5002 Meal). The test material was incorporated into the feed and mixed thoroughly to assure homogeneity. The test-material diet admixtures were prepared as fixed concentrations of test material. Fresh diets were prepared weekly. Prepared diets were covered and stored at room temperature following dispensing.

The homogeneous blend of the test material, prepared as a mixture in Certified Rodent Chow (5002 meal), was offered ad libitum to the treated rats of Groups 2, 3, 4 and 5. Control rats (Group 1) received Certified Rodent Chow (5002 meal) ad libitum only. Feed jars containing diet were replaced at least once each week. Animals had access to the test or control feeders -until the day of scheduled euthanasia.

Details on mating procedure:

The P1 mating period began after at least 10 weeks of P1 dosing and ended when all females were confirmed mated or approximately 3 weeks had elapsed. Each P1 male was assigned randomly (using animal reference numbers and a random numbers table) to be paired continuously with one P1 female of the same dose group to produce the F1 generation.

Mating was confirmed the morning following overnight pairing by observation of a copulatory plug (vaginal) and/or by the presence of sperm in a vaginal rinse. The day on which mating was confirmed was the female's Day 0 of gestation (GD 0). After confirmation of mating, each animal was returned to its own cage.

On GD 20, mated females were placed in clean cages fitted with a stainless steel litter pan and provided with fresh bedding material. Beginning on GD 21, mated females were examined at least twice daily for signs of parturition.

If a female was not confirmed mated, a litter pan was provided after examination on the last scheduled day of mating. Non-confirmed mated females were examined at least twice daily for signs of parturition after insertion of litter pans in their cages. One unconfirmed mated female was noted with red vaginal material approximately 2 weeks after overnight pairing. For husbandry purposes, this female was assumed to be at GD 14 and observations, body weight, and food consumption measurements were performed accordingly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material in feed was determined by the testing laboratory prior to and/or concurrent with dose initiation. Homogeneity was evaluated at two concentrations representing the lowest and highest levels expected during the study. Triplicate samples were collected from the top, middle, and bottom of each preparation (nine samples in total). The concentration was the mean of all nine samples.

Stability was assessed by measuring the concentrations of selected samples from each of the homogeneity preparations after room temperature and refrigerated storage.

Concentrations of test material-diet blends were checked by the testing laboratory at least once a month in order to assure continuing accuracy in mixing diets.

Concentration verification analysis indicated that all feed samples were within 13% of the nominal concentrations.

Duration of treatment / exposure:

The dosing regimen for all groups proceeded as follows: P1 males were dosed for at least 10 weeks prior to mating and through the mating period for F1 litters. P1 females were dosed for at least 10 weeks prior to mating, during the mating and gestation periods, and until they were euthanized following weaning of the F1 animals on PPD 21.

Details on study schedule:

P1 males selected for mating were euthanzied at the end of the mating period (Test Day 94). P1 females were euthanized after weaning of the F1 litters on PPD 21 (Test Day 121 or 133). Confirmed mated P1 females which did not give birth by presumed GD 26 or those females which had not been confirmed mated and did not give birth by 26 days after completion of the mating period, were euthanized and received gross necropsies. Special attention was paid to the reproduction system.

On PND 4, after counting, weighing, and examining the pups, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter. Partial adjustment (e.g., 5 males and 3 females) was permitted whenever there were not enough pups to obtain 4 per sex per litter. Litters of eight pups or less were not adjusted.

Culled pups were euthanized. Culled pups that appeared normal received only an external examination and tissues were not saved. Culled pups that appeared abnormal were subjected to a visceral examination.
At weaning (PND 21), the surviving neonates in each litter were group housed by sex until sacrificed on PND 28. On PND 28, after counting, weighing, and examining the neonates externally, all surviving F1 rats were sacrificed and discarded.

F1 litters were euthanized on PND 28.

Dose / conc.:

0

Remarks:

Group 1 (Control)

Dose / conc.:

0.25 other: %

Remarks:

Group 2

Dose / conc.:

0.5 other: %

Remarks:

Group 3

Dose / conc.:

0.75 other: %

Remarks:

Group 4

Dose / conc.:

1 other: %

Remarks:

Group 5

No. of animals per sex per dose:

10/sex/dose group

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

All animals were examined for viability at least once a day.

Male body weight was measured prior to P1 selection, on the first day of dosing (Day 0), and at least weekly thereafter until euthanized. Female body weight was measured prior to P1 selection, on the first day of dosing and at least weekly thereafter until confirmation of mating, then on Gestation Days (GD) 0, 7, 14, and 21 and on Postpartum Days (PPD) 0, 4, 7, 10, 14, and 21, and/or at least weekly until euthanized.

Food consumption was measured concurrently with body weight during the test period, except that food consumption was not measured during mating or following weaning of the F1 litters.

A clinical examination was given to each male prior to P1 selection, on the first day of dosing, and at least weekly thereafter until euthanized. Females received a clinical examination prior to P1 selection, on the first day of dosing, and at least weekly thereafter until confirmation of mating, then on GD 0, 7, 14, and 21, and on PPD 0, 4, 7, 10, 14, and 21.

Litter observations:

Dams were allowed to give birth. The duration of gestation was calculated and any difficulties occurring at parturition were noted. The date of parturition was recorded as the dam's Postpartum Day O (PPD 0).

Each morning and afternoon during the postnatal period, the litters were checked for dead offspring and unusual conditions, and the dams were examined for viability, nesting, and nursing behavior.

Dead pups were removed from the litter immediately after their discovery. If intact, dead pups were examined externally and internally for anomalies. Dead pups discovered on PND O also were examined internally to determine whether they were stillborn.

On PND 0, 1, 4, 7, 14, 21, and 28 the offspring were counted, sexed, and each live pup was weighed. Pups were counted and examined externally on a daily basis during the postnatal period.

Postmortem examinations (parental animals):

Gross necropsies were performed on all adult animals. Body weight was recorded on the day of necropsy. The uterus of each female used for mating, but failing to deliver, was examined grossly for evidence of implantations and these data were recorded.

Postmortem examinations (offspring):

All F1 pups that died were subjected to a visceral examination.

Statistics:

Bartlett's test of homogeneity of variance was used to determine if the groups have equivalent variances at the 1% level of significance. If the variances were equivalent, the hypothesis that there was no difference in response between the groups was tested using a standard one-way analysis of variance. If the ANOVA was significant, Dunett's test was performed to determine which treated groups differed from control. A linear regression to test for a dose response also was performed and tested for lack of fit.
If the variances were not equivalent, then a Kruskal-Wallis (non-parametric) test was performed to determine if the treatment effects are equivalent. If there was a difference, Dunn's Rank Sum comparison was used to determine which treatment groups differed from control. Jonckheere's test for ordered response also was performed .
Pup weight was analyzed by a standard nested analysis of covariance with pups nested within dams and with dams nested within doses, and litter size (both sexes combined) as the covariate. If differences in groups were identified, the Least Significant Difference (LSD) technique was used to determine which groups differed from the control group. Male and female pups were tested separately (the covariate was combined sexes in each analysis). All tests were reported at the 5% or 1% level of significance.
Parental reproductive and offspring survival incidence data were analyzed for statistical significance as follows: First, a standard chi-square analysis was performed to determine if the proportions of incidences differed between the groups tested. In keeping with standard statistical practice, if any one cell had an expected value less than 5, this step was not reported. Next, each treatment group was compared to the control group using a 2 x 2 Fisher Exact test. Thirdly, Armitage's test for linear trend in the dosage groups was performed.

All tests were reported at the 5% or 1% level of significance.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs which were judged to be directly related to treatment with MRD-94-775.

The majority of animals in all groups had no adverse clinical signs during the premating/mating, postmating, gestation, and/or postpartum periods.

There was a very low incidence of incidental findings observed in the male and/or female animals in one or more groups during the premating/mating, postmating, gestation, and/or postpartum periods.

These findings included dental abnormalities, scabs/sores, soft stool, alopecia, and/or ocular discharge. These findings were considered incidental and unrelated to treatment with the test substance.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were statistically significant lower mean body weights compared with controls in the 0.75% dose males (6-11%) and 1.0% dose males (n=-13%) from Day 21 through Day 70 during the premating period. These lower body weights occurred in a dose related manner. Similarly, there was statistically significant suppression in body weight gain in the 0.75% and 1.0% dose males during the Day 7/14, 14/21, and 28/35 intervals, and in the 1.0% dose males during the Day 42/49 interval compared with controls. Suppression in body weight gain also was observed during the remaining premating intervals in the 0.75% and 1.0% dose males, however these differences were not statistically significant.

In the females, there were no biologically significant changes in mean body weight or body weight change between treated and control animals. A statistically significant increase in mean body weight was observed in the 0.25% dose females when compared with controls at the Day 21 premating interval. This single occurrence was considered spurious and unrelated to treatment. There were several differences, both increases and decreases, in mean body weight gain between treated and control animals observed sporadically during premating. In the absence of a consistent pattern of response, these differences were considered incidental and unrelated to treatment .

During gestation, the mean body weight of the 1.0% dose females was statistically significantly lower(10-11 %) than controls at the GD 7, 14, and 21 intervals. Similarly, the mean body weight over the entire gestation period (GD 0-21) was lower than controls (14%), although this difference was not statistically significant. These lower body weights are due, at least in part, to the slightly lower body weights observed during the premating period which were evident at the start of gestation. There were no statistically significant differences in mean gestation body weight change between treated and control animals at any interval.

During the postpartum period, there was a dose-related decrease in body weight. Statistically significantly lower body weights were observed in the 1.0% dose females (9-23%) at every body weight interval and in the 0.75% dose females (12-14%) at every body weight interval except for PPD 0 when compared with controls. Statistically significant body weight gain suppression and/or body weight loss was observed in all treated groups at the PPD 0-4 interval, in the 1.0% dose females at the PPD 4-7 interval, and in the 0. 75% and 1.0% dose females over the entire postpartum interval (PPD 0-21).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the pre-mating period, dose-related decreases in mean food consumption were observed in male animals during the majority (Weeks 2-10) of the premating intervals and lower mean food consumption values compared with controls were observed in the 1.0% dose males during the majority of the study, although differences from controls were statistically significant only during Weeks 3 (8% ), 5 ( 10% ), and 9 ( 11%).

In the females, there were statistically significant lower food consumption in the 0.5% (9%), 0.75% (8.5%), and 1.0% {15%) dose groups during Week 7 and in 1.0% dose group {13%) during Week 9 compared with controls. Lower mean food consumption compared with controls was observed in I .0% dose females during the remaining premating intervals. However, these differences were not statistically significant.

During gestation, dose-related decreases in mean food consumption were observed in the female animals during the GD 0-7 and GD 7-14 intervals, as well as for the overall gestation period (GD 0-21 ). Statistically significant lower mean food consumption values when compared with controls were observed in the I .0% dose females during the GD 0-7 (18%) and GD 7-14 ( 18%) intervals, as well as the overall gestation period (17%).

During the postpartum period, dose-related decreases in mean food consumption were observed in female animals at every interval, including the overall postpartum period {PPD 0-21). Statistically significant lower mean food consumption values when compared with controls were observed in the 0.5% dose females at the PPD 0-4, 7-10, 10-14 and 14-21 intervals {16-30%), in the 0.75% dose females at the PPD 4-7, 7-10, 10-14 and 14-21 intervals(21-25%),and in the 1.0% dose females at every postpartum interval {37-41%). For the overall postpartum interval (PPD 0-21), statistically significant lower mean food consumption when compared with controls was observed in 0.5% (18%), 0.75% (24%) and 1.0% (40%) dose groups.

In the 0.25% dose females, statistically significant lower mean food consumption values compared with controls also were observed. However, these significant decreases were limited to the PPD 0-4 and PPD 4-7 intervals (18-28%). Mean food consumption during the overall postpartum interval (PPD 0-21) was not statistically significant lower than controls. Thus, the decreases observed in the 0.25% group during the early postpartum period were not considered biologically significant.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences in mean Male Fertility, Male Mating, Female Fertility, Female Fecundity, or Female Gestational Indices between treated and control animals. However, the mean Male Fertility (60%) and Female Fecundity (71.4%) indices of the 0.75% dose group was substantially lower than controls, lower than all other treated groups, and lower than the historical control range for this laboratory (Male Fertility 66.7-100%; Female Fecundity 72.2-100%). In the absence of a clear dose response or statistical significance, the biological importance of this finding is questionable.

Mean days of gestation of the treated and control groups were essentially equivalent.
There were no statistically significant differences in the mean percentage of live and dead offspring or in the sex ratio of the treated offspring compared with controls. One control female, three 0.25% dose females, one 0.5% dose female, four 0. 75% dose females, and two 1.0% dose females were not pregnant.

Dose descriptor:

conc. level: 0.75

Effect level:

0.75 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Food efficiency:

not examined

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

not examined

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related clinical findings observed in the offspring of any group. The majority of offspring in all groups were free of observable abnormalities from PND 0-21. Some offspring across most groups were observed without milk in their stomachs, during the first week of the postnatal period, with the highest incidence occurring on PND 0.
Single or low incidences of lacerations, scabs, alopecia, truncated tail, and/or missing limb were observed in one or more groups. These findings were considered incidental and unrelated to treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no statistically significant differences between treated and control offspring in any of the offspring survival indices.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly lower mean body weights compared with controls were observed in the treated offspring of the 0. 75% and 1.0% dose groups. In the male offspring, statistically significant lower mean body weight was observed in 0. 75% dose group on PND 14 (19%), PND 21 (20%), and PND 28 (15%), and in the 1.0% dose group on PND 0 (15%), PND 4 (22%), PND 7 (28%), PND 14 (35%), PND 21 (39%), and PND 28 (35%).

Similarly, in the female offspring, statistically significant lower mean body weight was observed in the 0.75% dose group on PND 14 (21 %), PND 21 (21%) and PND 28 (17%), and in the 1.0% dose group on PND 0 (12%), PND 7 (27%), PND 14 (36%), PND 21 (39%) and PND 28 (32%). These lower mean body weights were considered treatment-related.

There also were statistically significant lower mean body weights in the 0.5% dose males and females on PND 14 (14% and 12%, respectively) and 21 (14% and 12%, respectively). However, these values were essentially within the historical control range of this laboratory and thus were considered incidental. However, the PND 0 and 4 weights for both males and females were outside the historical control range for this laboratory, although not statistically significantly less than controls. This may be indicative of slight growth retardation.

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:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The majority of animals which died prior to scheduled termination (PND 0-20) were free of observable abnormalities or were too autolyzed/cannibalized to be examined at the necropsy. There were single incidences of dilated renal pelves, scabs, emaciated, anogenital staining, and/or apparent anophthalmia/anencephaly /protruding tongue. Two pups in the control group were stillborn and two pups in the 1.0% group were emaciated. Due to the very low incidence of findings, all were considered incidental and unrelated to treatment with the test material.

Histopathological findings:

not examined

Key result

Dose descriptor:

conc. level:

Generation:

F1

Effect level:

1 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

conc. level:

Generation:

F1

Effect level:

0.75 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

conc. level:

Generation:

F1

Effect level:

0.5 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: PND 0 and 4: Not statistically significant different from control, but out of historical control range. Indication of growth retardation

Conclusions:

Based on the study conditions, signs of toxicity were apparent at dose levels of 0.75% and 1.0%, and were observed in both the parental animals and offspring. Signs of toxicity in the parental animals included decreased body weight, suppression in body weight gain, and decreased food consumption. In the 0.5% dose group, adverse findings were limited primarily to decreases in food consumption compared to controls in the females during the postpartum period. Similarly, body weight reductions were observed in offspring of the 0.75% and 1. 0% dose groups of both sexes. There was also slight evidence of growth retardation in the 0.5% offspring since the PND 0 and 4 weights were outside historical control range for this laboratory (although not statistically significantly less than controls). Based on these results, 0.8% was selected as the high dose for the definitive two-generation reproduction toxicity study in rats with the test substance. This dose was anticipated to produce signs of toxicity, primarily lower body weights, in the parental males but also in the females during gestation and lactation. Additionally, this dose was considered low enough to allow for sufficient survivorship in the F1 generation. A low dose of 0.2% was selected because it was expected to be a level without effect, particularly in the F2 generation. Finally, 0.4% was selected as the mid dose.

Executive summary:

This study was designed to provide general information in order to select dose levels for a subsequent definitive multigenerational study with the substance 1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich. The study was conducted in compliance with the OECD Principles of Good Laboratory Practice. This study did not follow any specific regulatory testing guidelines. Per study schedule, P1 males and females received test material daily, via the diet, for at least ten weeks prior to mating and during the mating period. Additionally, P1 female animals received test material during the gestation and postpartum periods, until weaning of the F1 offspring on Postpartum Day (PPD) 21.

Clinical in life observations, body weight, and food consumption were recorded for all P1 animals at least weekly during the premating and mating periods (food consumption was not measured during mating due to cohabitation), and for females on Gestation Days (GD) 0, 7, 14, and 21 and on Postpartum Days (PPD) 0, 4, 7, 10, 14, and 21. Following birth, the offspring were counted and examined externally daily from Postnatal Day (PND) 0 to 21 and on PND 28. Offspring were sexed and weighed on PND 0, 1, 4, 7, 14, 21 and 28. P1 males were euthanized at the end of mating (Test Day 94), while females were euthanized following weaning of their litter son PPD21 (Test Day 121 or 133). A gross necropsy was performed on all adult animals and on all animals which cited during the study. A full macroscopic examination was performed on these animals.

All parental animals survived to scheduled terminal sacrifice and there were no clinical signs which were judged to be directly related to treatment with the test substance. The majority of animals in all groups had no adverse clinical signs during the premating/mating, postmating, gestation, and/or postpartum periods. Statistically or biologically significant lower mean body weight and corresponding suppression in body weight was observed in the 0.75% and 1.0% males compared with controls during the majority of the premating period. In the females, statistically significantly lower mean body weights compared with controls were observed in the 1.0% treated females during the gestation and postpartum periods and in the 0.75% treated females during the postpartum period.

Slight decreases in mean food consumption were observed in the 1.0% treated males and females during the majority of the premating period. Additionally, statistically significant decreases in mean food consumption were observed in the 1.0% treated females compared with controls during gestation. During the postpartum interval, consistent decreases in mean food consumption were observed in the 0.5%, 0. 75%, and 1.0% treated groups compared with controls. It is important to note that during the postpartum period test material consumption is substantially increased due to increased food consumption in the lactating dams. Thus, the animals in the treated groups are actually receiving higher doses than the dose rate at the end of the premating period.

Lower body weights were observed in offspring. The mean body weight of the 1.0% treated male and female offspring was statistically significantly lower than controls during the majority of the postnatal weighing intervals and all values were lower than the laboratory historical control range. In the 0.75% dose males and females, there were statistically significantly lower mean body weight during the postnatal (PND) 14, 21, and 28 intervals, and the mean body weight was lower than the laboratory historical control range on PND 0 and 4.

Based on this study conditions, signs of toxicity were apparent at dose levels of 0.75% and 1.0%, and were observed in both the parental animals and offspring. Signs of toxicity in the parental animals included decreased body weight, suppression in body weight gain, and decreased food consumption. In the 0.5% dose group, adverse findings were limited primarily to decreases in food consumption compared to controls in the females during the postpartum period. Similarly, body weight reductions were observed in offspring of the 0.75% and 1. 0% dose groups of both sexes. There also was slight evidence of growth retardation in the 0.5% offspring. The PND 0 and 4 weights were outside historical control range for the laboratory (although not statistically significantly less than controls) indicating possible growth retardation.

 

Based on these results, 0.8% was selected as the high dose for the definitive two-generation reproduction toxicity study in rats with the test substance. This dose was anticipated to produce signs of toxicity, primarily lower body weights, in the parental males but also in the females during gestation and lactation. Additionally, this dose was considered low enough to allow for sufficient survivorship in the F1 generation. A low dose of 0.2% was selected because it was expected to be a level without effect, particularly in the F2 generation. Finally, 0.4% was selected as the mid dose.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Remarks:

This study followed the study schedule outlined in OPPTS Guideline 870.3800 (EPA, 1994).

Adequacy of study:

supporting study

Study period:

Study Initiation: 11 July 1995
Inlife Test Period: 12 June 1995 to 7 April 1996
Completition Date: 31 October 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

July 1994

Qualifier:

equivalent or similar to guideline

Guideline:

other: Dangerous Substances Directives 67/548/EEC (Two-generation reproduction toxicity test)

Version / remarks:

1998

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA 40 CFR Part 798, Toxic Substance Control Act (Test Guideline for Reproduction and Fertility Effects)

Version / remarks:

1985

GLP compliance:

yes

Remarks:

This study was conducted in compliance with OECD (1981), EPA (1989) and EC 89/569/EEC (1989) Good Laboratory Practices.

Limit test:

no

Justification for study design:

The rat is among the species of choice for reproduction and fertility testing according to the E.C. Dangerous Substances Directive (67/548/EEC), Annex V part B "Two-Generation Reproduction Toxicity Test" (EC, 1988), and the U.S. EPA TSCA test guidelines for reproduction and fertility effects (EPA, 1985).

The dietary route is a preferred route of administration for non-volatile materials according to E.C. Dangerous Substances Directive Annex V, and U.S. EPA regulations. It also represents a likely route of human exposure.

This study report describes two satellite studies that were conducted during the definitive study. One study was designed to assess recovery in a group of satellite offspring by switching the control and high dose diet of the weanlings; the other study assessed the effects of the test material specifically on lactation and subsequent development by cross-fostering offspring from P1 dams in the control and high dose group.

Specific details on test material used for the study:

CAS number: 68515-49-1
Chemical name: 1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich
Internal Identification: MRD-94-775
Supplier: Exxon Chemical, Holland, BV
Date Received: April 8,1994
Expiration Date: April 1999
Description: Colorless liquid
Storage Condition: Room temperature
Purity: The test material was assumed 100% pure for the purpose of dosing

Species:

rat

Strain:

other: Crl:CDBR- VAF/Plus

Details on species / strain selection:

The rat is among the species of choice for reproduction and fertility testing according to the E.C. Dangerous Substances Directive (67/548/EEC), Annex V part B "Two-Generation Reproduction Toxicity Test" (EC, 1988), and the U.S. EPA TSCA test guidelines for reproduction and fertility effects (EPA, 1998).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. Kingston facility, Stone Ridge, New York (males) and Portage facility, Portage, Michigan (females)
- Age at study initiation: (P1) Males/Females: approximately 7-8 weeks
- Weight at study initiation: (P1) Males: 238.2-288.4 g; Females: 148.3-201.5 g
- Fasting period before study: No
- Housing: Individually housed during the test period, except during the mating and postpartum periods.
- Diet: Purina Certified Rodent Diet 5002 (Meal), ad libitum
- Water: Automatic watering system, ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40 to 70 percent relative humidity
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 12 June 1995 to 7 April 1996

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Mixing of feed: The basal diet consisted of Certified Rodent Chow (5002 Meal). The test material was incorporated into the feed and mixed thoroughly to assure homogeneity. The test material diet admixtures were prepared as fixed concentrations of test material.

- Fresh diets were prepared weekly. Prepared diets were covered and stored at room temperature following dispensing.

Details on mating procedure:

The P1 mating period began after at least 10 weeks of P1 dosing and ended when a female was confirmed mated or two weeks had elapsed. Each P1 male was assigned randomly (using animal reference numbers and a random numbers table) to be paired continuously with one P1 female of the same dose group to produce the F1 generation.

Mating was confirmed the morning following overnight pairing by observation of a copulatory plug (vaginal) and/or by the presence of sperm in a vaginal rinse. The day on which mating was confirmed was considered GD 0. After confirmation of mating, each animal was returned to its own cage.

On GD 20 (±1 day), mated females were single housed in clean cages fitted with stainless steel litter pans and provided with fresh bedding material. Beginning on GD 21, mated females were examined at least twice daily for signs of parturition.

If a female was not confirmed as mated, a litter pan was provided 20 days after the first day vaginal smears were evaluated for sperm. After the litter pan was added, these females were examined for signs of parturition at least twice daily until 26 days after the last day of mating had elapsed.

The mating procedures detailed above were the same for the P2 generation.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

- P1 males were dosed for at least 10 weeks prior to mating, through the mating period for F1 litters and until their sacrifice.
- P1 females were dosed for at least 10 weeks prior to mating, during the mating and gestation and postpartum periods, until sacrificed.
- The F1 male and female pups selected for the sorting population for the P2 adults were dosed from PND 21 through Day 0 of the P2 generation.
- P2 males and females followed the same dosing regimen as the P1 generation.

Frequency of treatment:

The homogeneous blend of the test material, prepared as a mixture in Certified Rodent Chow (5002 meal), was offered ad libitum to the treated rats. Control rats received Certified Rodent Chow (5002 meal) ad libitum. Feed jars containing diet were replaced at least once each week. Animals had access to the test or control feeders until the day of scheduled sacrifice.

Dose / conc.:

0 other: %

Remarks:

PMI Certified Rodent Diet 5002 only.

Dose / conc.:

0.8 other: %

Remarks:

Test item

No. of animals per sex per dose:

10/sex/dose group.

Control animals:

yes, plain diet

Details on study design:

Two satellite studies were conducted during the definitive study consisting of two groups of animals per study. One satellite study, the cross-fostering study, was conducted to differentiate between effects of in utero exposure vs. postnatal exposure on the offspring. This study also assessed recovery from in utero exposure. The second satellite study, the switched diet study, was conducted to assess the recovery of weanlings from in utero and postnatal exposure to the test material. This study also investigated the effect of the test material exposure on weanling rats. Two groups of 20 males, fed either control or high dose diets, served as mates for the satellite females.

PND 0 Cross-fostering:
On PND 0 of the F1 generation only, the pups from ten satellite Group 1 (control) litters and ten satellite Group 4 (0.8%) litters were switched (i.e. each litter of control pups was placed with one of the Group 4 dams and each litter of Group 4 pups was placed with a Group 1 dam). The litter sizes of each pair of dams being switched was as close to the same size as possible (i.e. total number of pups was the same 2 pups). The data from these animals was used to determine whether the Group 4 weights were being affected by the quality of milk, health of the dam or the size of the pup at birth. The data generated from these animals after this point was not used as part of the main study and these animals were not subject to selection for the next generation. All other procedures (i.e. observations, body weights, PND 4 culling, etc.) for the F1 generation were conducted on these pups. However, all pups from these litters surviving on PND 21 were allowed to become adults. These animals were treated in the same manner (i.e. observations, body weights, food consumption, etc.) as the main study P2 animals. These animals were sacrificed after the beginning of the mating period for the P2 generation and a gross postmortem examination was performed using the same procedure as the P1 adults.

PND 21 Switched Diet:
On PND 21 of the F1 generation only, all surviving pups in the Group 1 and Group 4 litters, that were not selected for the main study P2 generation, were allowed to become adults for the switch diet satellite study. All pups from the Group 4 females were fed control diet and the pups from the Group 1 females were fed Group 4 diet. These animals received their respective switched diets for the duration of the P2 premating period. The data generated from these animals after this point were not used as part of the main study and these animals were not subject to selection for the next generation. These animals were treated in the same manner (i.e. observations, body weights, food consumption, etc.) as the main study P2 animals.

Animals were sacrificed after the beginning of the P2 mating period and a gross postmortem examination was performed using the same procedure as the P1 adults. The necropsy of the satellite offspring (cross-fostered and switched diet) was performed in the same manner as the P1 adults. Satellite animals' organs were weighed and tissues preserved as listed for the P1 adults. Estrous cycle evaluation was conducted using the same procedure as the P1 adults

Parental animals: Observations and examinations:

All animals were examined for viability at least once a day. Cage-side observations were performed daily on all P1 adults, except the days on which clinical observations were performed. Cage-side observations also were performed after weaning for all F1 offspring selected to be considered for the P2 generation.
A clinical examination was given to each male prior to P1 selection, on the first day of dosing (Day 0), and at least weekly thereafter until sacrifice. Females received a clinical examination prior to P1 selection, on the first day of dosing, and at least weekly thereafter until confirmation of mating, then on GD 0, 7,14, and 21, and on PPD 0,4,7,10,14, and 21. A clinical examination also was given to each P1 male
and female on its day of sacrifice.
Male body weight was measured prior to P1 selection, on the first day of dosing, and at least weekly thereafter until sacrifice. Female body weight was measured prior to P1/P2 selection, on the first day of dosing and at least weekly thereafter until confirmation of mating, then on GD 0, 7, 14, and 21 and on PPD 0, 4, 7, 10, 14, and 21, and/or at least weekly until sacrifice. Body weight also was measured on the day of sacrifice for all P1 males and females.
Food consumption was measured concurrently with body weight after Day 0, except during mating or during the postweaning period of the F1 litters.
The inlife procedures detailed above were the same for the P2 generation.

Oestrous cyclicity (parental animals):

The estrous cycle of each P1 female was evaluated daily by vaginal smears beginning three weeks prior to mating (Day 49) and continuing until the end of cohabitation. The vaginal smears were stained with Wright's stain before being evaluated. Estrous cycles were not evaluated on the day positive vaginal smears for either sperm or plugs were present.
The estrous cycle evaluation detailed above was the same for the P2 generation.

Sperm parameters (parental animals):

Not examined.

Litter observations:

Each morning and afternoon during the postnatal period, the litters were checked for dead offspring and unusual conditions, and the dams were examined for viability, nesting, and nursing behavior.

Dead pups were removed from the litter immediately after their discovery and subjected to a gross necropsy if their condition permitted.

On PND 0,1,4, 7,14, and 21 the offspring were counted, sexed, and each live pup was weighed. Pups were counted and examined externally on a daily basis during the postnatal period.

On PND 4, after counting, sexing, weighing, and examining the pups, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter. Partial adjustment (e.g., 5 males and 3 females) was permitted whenever there were not enough pups to obtain 4 per sex per litter. Litters of eight pups or less were not adjusted. Culled pups were sacrificed. Culled pups received only an external examination and tissues were not saved.

The postnatal evaluations detailed above were the same for the F2 generation. At weaning (PND 21) of the F1 generation only, the offspring were examined externally and two offspring/sex/litter were selected randomly and group housed by sex to be considered for P2 generation adults. These offspring were observed and weighed on a weekly basis during the interim period from the first PND 21 to the time the last delivered litter reached PND 21.

From the remaining F1 offspring and all surviving F2 offspring on PND 21, one offspring/sex/litter in each group was selected randomly, sacrificed and examined for internal abnormalities. All remaining F1 offspring from Groups 2 and 3, and all remaining F2 offspring were examined externally and sacrificed. Offspring that appeared normal externally were not subjected to further examination and no tissues were saved. Offspring that appeared abnormal externally were subjected to an internal examination at the discretion of the Study Director or his designee. All remaining F1 offspring for Groups 1 and 4 became the Switched Diet satellite animals.

Beginning on PND 29, all surviving F1 female offspring were examined daily for vaginal opening. Beginning on PND 45, all surviving F1 male offspring were examined weekly for preputial separation. The examinations continued until all animals reached criteria (i.e. vaginal opening or preputial separation). If all females did not reach criteria before Day 0 of the F2 generation, the examinations continued only on the animals which became the P2 generation. All extra F1 offspring were sacrificed on Day 0 of the P2 generation regardless of reaching criteria. Only data for the offspring which became P2 adults was reported.

Postmortem examinations (parental animals):

SACRIFICE
- All animals (adults and offspring) were sacrificed by CO2 asphyxiation and exsanguination.
- All P1 and P2 males were sacrificed after the mating of the satellite females. P1 and P2 females were sacrificed after weaning of their litters. Confirmed mated females which did not give birth by presumed GD 26, or those females which had not been confirmed mated and did not give birth by 26 days after the last day of mating were sacrificed and received gross necropsies. Special attention was paid to the reproductive system. Any female whose entire litter succumbed or was euthanized was sacrificed after the last offspring succumbed.

GROSS NECROPSY
- Gross necropsies were performed on all adult animals, including those that were found dead or sacrificed. Body weight was recorded on the day of necropsy. The uterus of each female used for mating was examined grossly for evidence of implantations and those data were recorded. Additionally, the corpora lutea were counted and the number counted for each ovary were recorded (total count for the animal reported).

Postmortem examinations (offspring):

SACRIFICE
- All animals (adults and offspring) were sacrificed by CO2 asphyxiation and exsanguination.
- All P1 and P2 males were sacrificed after the mating of the satellite females. P1 and P2 females were sacrificed after weaning of their litters. Confirmed mated females which did not give birth by presumed GD 26, or those females which had not been confirmed mated and did not give birth by 26 days after the last day of mating were sacrificed and received gross necropsies. Special attention was paid to the reproductive system. Any female whose entire litter succumbed or was euthanized was sacrificed after the last offspring succumbed.

GROSS NECROPSY
- Gross necropsies were performed on all adult animals, including those that were found dead or sacrificed. Body weight was recorded on the day of necropsy. The uterus of each female used for mating was examined grossly for evidence of implantations and those data were recorded. Additionally, the corpora lutea were counted and the number counted for each ovary were recorded (total count for the animal reported).

Statistics:

The following parameter went though statistical significance tests: pup weight, parental reproductive and offspring survival incidence; spermatoxicity and oocyte count; estrous cycle; and development landmarks.

Offspring viability indices:

Offspring survival

Clinical signs:

no effects observed

Description (incidence and severity):

CROSS FOSTERING SATELLITE - PARENTAL RESULTS - P1 CROSS-FOSTERING DAMS
There were no clinical signs judged to be directly related to treatment with the test material in the dams which were cross-fostering litters during the postpartum period. The majority of dams in both groups had no adverse clinical signs and observations were limited to incidental dental abnormalities and alopecia.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

CROSS FOSTERING SATELLITE - PARENTAL RESULTS - P1 CROSS-FOSTERING DAMS
The cross-fostered high dose adult females had statistically significant lower mean food consumption during the PPD 7/10, 10/14, and 14/21 intervals (up to 16%) and the overall postpartum period (10%) compared with the cross-fostered controls. These differences were considered biologically significant and consistent with the pattern observed in the main study animals during the postpartum period. There were no statistically significant suppressions in body weight gain between treated and control groups, although the cross-fostered high dose females body weight gain was 22% less than controls during the overall postpartum period.

Food efficiency:

no effects observed

Description (incidence and severity):

CROSS FOSTERING SATELLITE - PARENTAL RESULTS - P1 CROSS-FOSTERING DAMS
Mean measured dose rate of the cross-fostered high dose females was essentially
equivalent to the main study animals

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

CROSS-FOSTERING SATELLITE: P2 ADULT EQUIVALENTS RESULTS - P2 CROSS-FOSTERED ADULTS
During the premating period, the majority of P2 equivalents in both groups had no adverse clinical. There was a very low incidence of incidental observations including dental abnormalities, scabs/sores, necrotic/truncated tail, alopecia, ocular discharge, nasal discharge, enlarged eye, dyspnea, emaciation, little sign of food consumption, little sign of stool, red material around penis, and/or swollen snout. All observations were considered incidental and unrelated to treatment with the test material.

The majority of cross-fostered offspring of both groups were free of abnormalities at postmortem examination. The most notable postmortem observation was a low incidence of dilated renal pelves in males and females cross-fostered with control or high dose dams. Incidental observations included dental abnormalities, truncated tail, alopecia, ocular discharge, enlarged eye, discolored kidney or thymus, flaccid kidney, small prostate or seminal vesicles, large mandibular lymph nodes or thymus, an enlarged thickened liver with accessory lobes, abnormal spleen, distended uterus, and/or urinary bladder abnormalities. None of these observations were considered treatment-related due to the low, infrequent incidence. One male offspring cross-fostered with a control dam was sacrificed for humane reasons on Day 29 with severe dental abnormalities which resulted in poor food consumption and sores of the palate. One female offspring cross-fostered with a control dam was found dead on Day 2 with urinary bladder and kidney abnormalities. These limited occurrences were not considered treatment-related.

SWITCHED DIET OFFSPRING SATELLITE: P2 ADULT EQUIVALENTS
During the premating period, the majority of switched diet P2 equivalents in both groups had no adverse clinical signs. There was a very low incidence of incidental observations including dental abnormalities, scabs/sores, alopecia, ocular discharge, anogenital staining, wet rales and emaciation. All observations were considered incidental and unrelated to treatment with the test material.

The majority of switched diet P2 equivalents were free of abnormalities at postmortem examination. There was a low incidence of dilated renal pelvis in males which was observed in both the control and high dose switched diet P2 equivalents. Incidental postmortem observations included a very low incidence of dental abnormalities, alopecia, ocular discharge, sores palate, scabs, small prostate, and/or urinary bladder abnormalities. No observation was considered treatment-related due to the low, sporadic incidence.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

CROSS-FOSTERING SATELLITE: P2 ADULT EQUIVALENTS RESULTS - P2 CROSS-FOSTERED ADULTS
During the entire premating period, the mean body weight of the offspring cross-fostered with high dose dams continued to be statistically significantly lower (9-11 % males; 7-10% females) than the mean body weight of the offspring cross-fostered with control dams. Similarly, the mean body weight gain of the offspring cross-fostered with high dose dams was lower than the offspring cross-fostered with control dams at the majority of intervals. However, these differences were not always statistically significant.

Compared to the main study control offspring, the body weights of male and female offspring cross-fostered with control dams were statistically significantly increased compared to the main study animals during the early part of the premating period (males: Day 0-14; females: Days 0-28). This trend was consistent with the two week postweaning intervals and was probably due to the fact that the cross-fostered offspring were the oldest offspring (they were from the first two days of deliveries) and therefore the heaviest, while the main study control offspring came from a variety of delivery dates covering a period of almost two weeks and their postweaning weights represent animals of mixed ages. By Day 21, the mean body weight of the offspring crossfostered with control dams and main study control offspring were essentially equivalent (< 6% difference) in both sexes. Near the end of the P2 premating period, the weights of main study
control males became greater than the cross-fostered control males. The difference between the main study control males and the cross-fostered control males began to widen during the last two weeks of the P2 premating period. However, these differences were small (<4%) and not statistically significant.

The mean body weight of offspring cross-fostered with high dose dams was comparable to the main study controls during the early part of premating. However, during the later part of premating, the mean body weight of the offspring cross-fostered with high dose dams was statistically significantly decreased compared to the main study control offspring of both sexes (males: Days 21-70; females Days: 35-70). Similarly, mean body weight gain of the offspring cross-fostered with high dose dams was lower at all premating intervals (except Day 49/56 in the females).

SWITCHED DIET OFFSPRING SATELLITE: P2 ADULT EQUIVALENTS
There were no statistically significant differences in mean body weight of the switched diet control or high dose male P2 equivalents during the entire premating period. The mean body weight of the female switched diet high dose P2 equivalents was statistically significantly lower than the female switched diet control P2 equivalents throughout most of premating. However, these differences were small (<10%) and not considered biologically important. There were no biologically important differences in mean body weight change between the switched diet control or high dose animals of either sex during premating.

The switched diet control male body weights were statistically significantly lower than the main study control body weights on Days 21,42,49, 56, 63 and 70. By Days 63 and 70 the switched diet control male body weights were approximately 10% less than the main study control. No particular importance was assigned to this difference, i.e., it maybe related to normal biological variation. The females of the switched diet control did not show this effect and their mean body weights were equivalent to the main study control throughout the premating period. The comparison of the main study control and the switched diet high dose group revealed statistically significant lower body weights in both sexes for the majority of the intervals. By Day 70 of the premating period, the difference between the switched diet high dose males and females compared with main study controls was 9.6% and 7.2%, respectively.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

CROSS-FOSTERING SATELLITE: P2 ADULT EQUIVALENTS RESULTS - P2 CROSS-FOSTERED ADULTS
The mean food consumption of the offspring cross-fostered with the high dose dams was statistically significantly lower than the offspring cross-fostered with control dams at the majority of the 10 week premating intervals in both sexes. However, these differences were small (<7%), and thus were not considered biologically significant. The mean food consumption of the main study control offspring and both the offspring cross-fostered with high dose dams or control dams were generally comparable during the premating period. There were, however, several statistically significant differences observed sporadically throughout the period. In the absence of a clear consistent pattern of response, these small (<9%) differences were not considered biologically important.

Mean measured dose rate of the offspring cross-fostered with high dose dams (males: 901-484 mg/kg/day; females: 894-572 mg/kg/day) was essentially equivalent to the main study offspring (males: 929-482 mg/kg/day; females: 927-561 mg/kg/day) in both sexes.

SWITCHED DIET OFFSPRING SATELLITE: P2 ADULT EQUIVALENTS
There were no statistically significant differences in mean food consumption between the switched diet control and high dose P2 equivalents of either sex. Similarly, there were no statistically significant differences in mean food consumption of the switched diet control or high dose P2 equivalents compared with the main study control P2 animals of either sex with the exception of a two small increases in the switched diet females during Weeks 6 (control) and Week 8 (high dose). These small (6%) differences were not considered biologically important.

Mean measured dose rate of the switched diet high dose P2 equivalents (males: 958-
496 mg/kg/day; females: 984-583 mg/kg/day) was essentially equivalent to the main study high dose P2 animals (males: 929-482 mg/kg/days; females: 927-561 mg/kg/day) in both sexes.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine 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, non-treatment-related

Description (incidence and severity):

CROSS-FOSTERING SATELLITE: P2 ADULT EQUIVALENTS RESULTS - P2 CROSS-FOSTERED ADULTS
There were statistically significant increases in the mean absolute and relative liver weights of the offspring cross-fostered with the high dose dams (males: 11% and 23%, respectively; females: 22% and 35%, respectively) compared with the offspring crossfostered with control dams. In the kidneys, there were statistically significant increases in the mean absolute and relative weights of the offspring cross-fostered with the high dose dams (males: 16% and 30%, respectively; females: 9% and 22%, respectively) compared with the offspring cross-fostered with control dams. These differences were very similar to the differences seen in the main study P2 generation, and similarly were regarded as not biologically significant.

There were statistically significant increases in the offspring cross-fostered with high dose dams mean absolute and relative adrenal weights in the females, mean relative right and left ovary weights in females, mean relative uterus weight in the females, and mean relative adrenal weight in the males; mean absolute and relative thymus weights of both males and females, mean absolute spleen weight in the males, both left and right testis weights in males, mean relative right testis and epididymis weights in the males, and mean relative brain weights of both sexes compared with the offspring cross-fostered with control dams. The exact cause of these weight differences was unknown, and since histopathology was not performed, it could not be determined if changes in tissue structure and function occurred. However, the majority of changes were probably compensatory changes related to decreased mean body weights of the offspring cross-fostered with high dose dams. Based on the histopathological results of the main study P2 adults, these effects were not considered biologically significant.

SWITCHED DIET OFFSPRING SATELLITE: P2 ADULT EQUIVALENTS
There were statistically significant increases in the mean absolute and relative liver weights of the switched diet high dose P2 equivalents (males: 36% and 34%, respectively; females: 31% and 39%, respectively) compared with the switched diet control P2 equivalents. In the kidneys, there were statistically significant increases in the mean absolute and relative weights of the switched diet high dose P2 equivalents (males: 27% and 27%, respectively; females: 15% and 23%, respectively) compared with the switched diet control P2 equivalents. These differences were very similar to the differences seen in the main study P2 generation, and similarly were regarded as not biologically significant.

There were no other differences in the mean absolute or relative organ weights which were considered biologically significant. There were, however, statistically significant increases in the switched diet high dose P2 equivalents' mean absolute brain weight in males, mean relative brain weight in females, mean absolute and relative adrenal weights in both males and females, mean absolute right and left testis weight and epididymis weights in males, and mean relative left testis and right epididymis weight in males compared with switched diet control P2 equivalents. The exact cause of these weight differences was unknown, and since histopathology was not performed, it could not be determined if changes in tissue structure and function occurred. However, the majority of changes observed in the females were probably related to the statistically significant decrease in mean body weight of the switched diet high dose P2 equivalents at study termination.

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

CROSS-FOSTERING SATELLITE: P2 ADULT EQUIVALENTS RESULTS - P2 CROSS-FOSTERED ADULTS
There was a statistically significant increase in mean estrous cycle length in the offspring cross-fostered with both the control (4.23 days) and high dose (4.38 days) dams compared with the main study P2 control females (4.18 days). However, this decrease was small (<5%), within the commonly reported estrous cycle lengths of 4-5 days for the rat and thus, not considered biologically significant.

There was not a statistically significant difference in the number of females with abnormal estrous cycle between offspring cross-fostered with either controls or high dose females and the main study P2 control females.

SWITCHED DIET OFFSPRING SATELLITE: P2 ADULT EQUIVALENTS
There were no statistically significant differences in mean estrous cycle length or animals with abnormal estrous cycles between the main study P2 controls and either the control or high dose switched diet P2 equivalents.

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

CROSS-FOSTERING SATELLITE: OFFSPRING RESULTS (PND 0-21) - F1 CROSS-FOSTERED OFFSPRING
The majority of offspring in both cross-fostered groups were free of observable abnormalities from PND 0-21. Some offspring in both groups were observed without milk in their stomachs primarily on PND 0. Truncated tail or hind legs was observed in two offspring on PND 0 and 1 cross-fostered to a control dam and in one offspring on PND 2-4 cross-fostered to a control dam. These observations were considered incidental and unrelated to treatment.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

CROSS-FOSTERING SATELLITE: OFFSPRING RESULTS (PND 0-21) - F1 CROSS-FOSTERED OFFSPRING
There were no statistically significant differences in the Live Birth, Day 1, Day 4, Day 7, Day 14, or Day 21 survival indices in the offspring cross-fostered with high dose dams compared to either the offspring cross-fostered with control dams or the main study controls. Likewise, there were no differences between the offspring cross-fostered with control dams compared with the main study controls. However, there were statistically significant increases in the Viability at Weaning Index for both cross-fostered groups (100%) compared with main study control (93.4%). These data support the conclusion that the cross-fostering dams nurtured the offspring as if they were their own offspring.

Three female and two male offspring cross-fostered to one control dam were found dead on PND 1 with no observable abnormalities. An additional male cross-fostered to another control dam was found dead on PND 1 and one male cross-fostered to a high dose dam was found dead on PND 2 with no observable abnormalities

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

CROSS-FOSTERING SATELLITE: OFFSPRING RESULTS (PND 0-21) - F1 CROSS-FOSTERED OFFSPRING
On PND 0 and 1, the mean body weights of the male offspring cross-fostered to high dose dams was significantly (p 0.05) greater (7% and 9%, respectively) than the male offspring cross-fostered to the control dams. By PND 4 and until PND 21, the mean body weights of the male offspring cross-fostered to the control dams exceeded (up to 14%) the offspring cross-fostered to the high dose dams with the differences being statistically significant on PND 14 and 21. The female offspring followed a similar trend, except that differences were statistically significant on PND 4, 14, and 21.

There were no statistically significant differences between the mean body weights of the offspring cross-fostered to control dams and the main study control offspring of either sex during the postnatal period. However, the mean body weights of the offspring cross-fostered to the high dose dams were statistically significantly lower (up to19%) than the main study control offspring of both sexes on PND 14 and 21.

CROSS-FOSTERING SATELLITE: POSTWEANING OFFSPRING RESULTS - F1 CROSS-FOSTERED OFFSPRING
During the two weeks postweaning (prior to the premating period), the mean body weight of the offspring cross-fostered with control dams was significantly (p 0.01) greater than the offspring cross-fostered with the high dose dams. This was consistent with the trend observed during the PND 14 and 21 intervals. However, the differences between the control and high dose decreased as the study progressed.

Compared to the main study control offspring, the body weight of male and female offspring cross-fostered with control dams were statistically significantly increased compared to the main study animals during the two weeks postweaning. However, this was probably due to the fact that the cross-fostered offspring were the oldest offspring (since they were from the first two days of deliveries) and therefore the heaviest, while the main study control offspring came from a variety of delivery dates covering a period of almost two weeks and the postweaning weights represent animals of mixed ages. This trend was not apparent when the mean body weight of offspring crossfostered with high dose dams was compared with the main study controls during Week 1 (weights were essentially equivalent). During Week 2 however, the mean body weight of the offspring cross-fostered with high dose dams exceeded the main study controls in both sexes.

SWITCHED DIET OFFSPRING SATELLITE: POSTWEANING OFFSPRING RESULTS-F1
During the two weeks postweaning (prior to the premating period), there were no statistically significant differences in mean body weight of the switched diet control or high dose offspring of either sex. The mean body weights of the switched diet control (both males and females) and high dose offspring (males only) were statistically significantly lower than the main study control offspring in the male and female animals during Week 1 only. By Week 2, the mean body weights were essentially equivalent.

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, non-treatment-related

Description (incidence and severity):

CROSS-FOSTERING SATELLITE: POSTWEANING OFFSPRING RESULTS - F1 CROSS-FOSTERED OFFSPRING
There were no statistically significant differences for day of preputial separation between the offspring cross-fostered with the control or high dose dams compared with the control offspring from the main study. Although the evaluations were conducted in accordance with the procedures listed in the September 1993 draft of the EPA Health Effects Test Guideline OPPTS 870.3800, the results of these evaluations were not meaningful because the frequency of the evaluations (weekly) was not sufficient to detect an effect.

In the females, the offspring cross-fostered with the control dams (35.7 days) and with the high dose dams (34.9 days) exhibited a statistically significant later maturation for vaginal patency compared with control offspring from the main study (32.2 days). However, this delay was small (< 4 days) compared with controls, and thus, not considered biologically significant.


SWITCHED DIET OFFSPRING SATELLITE: POSTWEANING OFFSPRING RESULTS-F1
In the females, switched diet control offspring (33.4 days) and high dose offspring (34.8 days) exhibited a statistically significant later maturation for vaginal patency compared with control offspring from the main study (32.2 days). However, this delay was small (< 3 days) compared with controls, was most likely secondary to the slightly lower body weight difference in the high dose compared with controls, and thus, not considered biologically significant.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Conclusions:

This study report describes two satellite studies conducted during the definitive study.

In the cross-fostered study, dams and offspring selected from the P1 and F1 generations on PPD/PND 0 to differentiate between effects of in utero exposure vs. postnatal exposure on the offspring. This study also assessed recovery from in utero exposure. In the switched diet study, offspring selected from the F1 generation on PND 21 which assessed for the recovery of weanlings from in utero and postnatal exposure to the test material. This study also investigated the effect of the test material exposure on weanling rats.

Results from the cross-fostering and switched diet satellite groups support the conclusion that exposure of lactating maternal animals to the test substance results in suppression of body weight gain in pups. In the switched diet phase, weanlings from high dose animals given control diet displayed signs of recovery in body weight immediately after weaning and the control weanlings switched to high dose diet displayed the same pattern of growth as the P1 generation high dose animals.

Executive summary:

A cross-fostering satellite study was conducted to assess the influence of postnatal exposure of the offspring (e.g. via lactation) on the body weight gain suppression observed in the main study. On PND 0 of the F1 generation only, the pups from ten satellite Group 1 (control) litters and ten satellite Group 4 (0.8%) litters were switched (i.e. each litter of control pups was placed with one of the Group 4 dams and each litter of Group 4 (0.8%)  pups was placed with a Group 1 dam. The litter sizes of each pair of dams being switched was as close to the same size as possible. The data from these animals was used to determine whether the Group 4 (0.8%) offspring body weight reductions resulted from postnatal events possibly including quality of milk, health or behaviour of the dam or direct exposure to the test material.

 

A switched diet satellite study was conducted to assess the recovery of weanlings from postnatal exposure to the test material, and the effect of the test material exposure on weanling rats. On PND 21 of the F1 generation only, all surviving pups in the Group 1 (control) and Group 4 (0.8%)  litters were allowed to become adults. All pups from Group 4 (0.8%) were fed control diet and the pups from Group 1 (control) were fed Group 4 (0.8%) diet. These animals received their respective switched diets for the duration of the P2 premating period.

 

Results from the cross-fostering and switched diet satellite groups support the conclusion that exposure of lactating maternal animals to the test substance results in suppression of body weight gain in pups. In the switched diet phase, weanlings from high dose animals given control diet displayed signs of recovery in body weight immediately after weaning and the control weanlings switched to high dose diet displayed the same pattern of growth as the P1 generation high dose animals.

 

In the cross-fostering satellite study, offspring born to high dose dams and cross-fostered to control dams on PND 0 exhibited body weights which were not different from main study control offspring throughout the postnatal phase. Conversely, the mean body weights of the offspring born to control dams and cross-fostered to the high dose dams were statistically significantly lower (up to 19%) than the main study control offspring of both sexes on PND 14 and 21 and continued to be statistically significantly lower (9-11% males; 7-10% females) than the mean body weight of the offspring cross-fostered with control dams during premating.

 

These observations support the conclusion that the offspring effects were due to an effect during lactation. Based on these findings, an Offspring NOAEL was established at 0.4% in the definitive study.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2001

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Cited as Directive 67/548/EEC, Annex V Part B 1988

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Principles of method if other than guideline:

-

GLP compliance:

yes

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

Molecular formula:

C28 H46 O4

IUPAC Name:

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

Constituent 2

Reference substance name:

1,2-benzenedicarboxylic acid, di-C9,C10 and C11 branched alkyl ester, C10 Rich

IUPAC Name:

1,2-benzenedicarboxylic acid, di-C9,C10 and C11 branched alkyl ester, C10 Rich

Details on test material:

- Name of test material (as cited in study report): 1,2-benzenedicarboxylic acid, di-C9, C10 and C-11 branched alkyl ester, C10 rich
- Physical state: liquid
- Analytical purity: Assumed 100% pure for purposes of dosing
- Expiration date of the lot/batch: April 1999

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 Laboratories, Inc
- Age at study initiation: (P) 7-8 wks
- Weight at study initiation: (P) Males: 238.2-288.4 g; Females: 148.3-201.5 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: No
- Housing: individually housed during the test period, except during the mating and postpartum periods.
- Diet: Purina Certified rodent Chow (5002 meal), ad libitum
- Water: Automatic watering system, ad libitum
- Acclimation period:16 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 40-70
- Photoperiod: 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: 1995-07-12 To: 1996-04-07

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): The basal diet consisted of Certified Rodent Chow (5002 Meal). The test material was incorporated into the feed and mixed thoroughly to assure homogeneity. The test material diet and mixtures were prepared as fixed concentrations of test material.

Details on mating procedure:

After the 10-week premating exposure period, each P1 male was randomly paired with a P1 female from the same treatment group to produce the F1 generation. The mating period ended when all females were confirmed mated or approximately two weeks had elapsed. The day of confirmation of mating, based on observation of a copulatory plug or sperm in a vaginal rinse, was designated as Gestation day 0 (GD 0) and the date on which parturition was recorded was designated as PND 0.

- M/F ratio per cage: 1:1
- Length of cohabitation: Continuously until mating was confirmed
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Mated females were single housed in clean cages fitted with a stainless steel litter pans and provided with fresh bedding material.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of test material-diet blends were checked by the testing laboratory at least once a month in order to ensure continuing accuracy in mixing diets.

Duration of treatment / exposure:

P1 males and females received test material daily for at least 10 weeks prior to mating and during the mating period. Additionally P1 females received test material during the gestation and postpartum periods, until weaning of the F1 (offspring of the P1 generation) offspring on PPD 21. P2 (F1 generation animals chosen to mate) males were dosed from postnatal day (PND) 21 for at least 10 weeks prior to mating, through the mating period for F2 (the offspring of the P2 generation) litters, and until sacrificed. P2 (F1) females were dosed from PND 21 for at least 10 weeks prior to mating, during mating, gestation, postpartum and until they were sacrificed following weaning of the F2 animals on PPD 21.

Frequency of treatment:

continuous (diet)

Details on study schedule:

- Selection of parents for F1 generation when pups were 21 days of age.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

0.2% dietary exposure

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

0.4% dietary exposure

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

0.8% dietary exposure

Doses / concentrations 4

Remarks:

Doses / Concentrations:
In the first study 0.2, 0.4, 0.8% were target dietary concentrations, 30/sex/group. In the second study the target concentrations were 0.02%, 0.06%, 0.2%, and 0.4% in diet, 30/sex/group.
Basis:
nominal in diet

No. of animals per sex per dose:

Main Study (P1 Generation)
30/sex/dose for 0.2% and 0.4% concentrations
40/sex/dose for control and 0.8% concentration

Satellite Animals (P1 Generation)
5 males/dose for 0.2% and 0.4% doses
10/sex/dose for control and 0.8% dose

Main Study (P2 Generation)
30/sex/dose

Satellite animals (P2 generation)
5 males/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Doses for this study were selected based on the results of a one-generation reproduction toxicity range-finding study in rats with the test material.

The dose levels tested in the rangefinding study (with only a 3 week pre-mating period) were 0.25%, 0.50%, 0.75% and 1.0%. Signs of toxicity were apparent at dose levels of 0.75% and 1.0%, and observed in both the parental animals and offspring. Signs of toxicity in the parental animals included decreased doby weight, suppression of body weight gain, and/or decreased food consumption. In the 0.5% dose group, adverse findings were limited primarily to decreases in food consumption compared to controls in the females during the postpartum period. Overt signs of toxicity observed in the offspring were limited to growth retardation in males and females at 0.75% and 1.0%. There also was slight evidence of growth retardation at 0.5%. The postnatal day 0 and 4 offspring mean body weights were outside the historical control range of this laboratory (although not statistically significantly less than controls) indicating possible growth retardation.

Based on these results, 0.8% was selected as the high dose for the definitive two-generation reproduction toxicity study in rats with the test material. This dose was anticipated to produce signs of toxicity, primarily lower body weights, in the parental males but also in the females during gestation and postpartum. Additionally, this dose was considered low enough to allow for sufficient survivorship in the F1 generation. A low dose of 0.2% was selected because it was expected to be a level without effect, particularly in the F2 generation. Finally, 0.4% was selected as the mid dose.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males: On the first day of dosing (day 0) and at least weekly therafter until sacrifice. Females: Prior to P1 selection, on the first day of dosing, and at least weekly thereafter until confirmation of mating, then on GD 0, 7, 14, and 21, and on PPD0, 4, 7, 10, 14, and 21. An exam was also given to each P1 male and female on its day of sacrifice.


BODY WEIGHT: Yes
- Time schedule for examinations: Males: prior to P1 selection, on the first day of dosing, and at least weekly thereafter until sacrifice. Females: Prior to P1/P2 selection, on the first day of dosing and at least weekly thereafter until confirmation of mating, then on GD 0, 7, 14, and 21 and on PPD 0, 4, 7, 10, 14, and 21, and/or at least weekly until sacrifice. Body weight also was measured on the day of sacrifice for all P1 males and females.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured concurrently with body weight after Day 0, except during mating or during the postweaning period of the F1 litters.

Oestrous cyclicity (parental animals):

The estrous cycle of each P1 female was evaluated daily by vaginal smears beginning three weeks prior to mating (day 49) and continuing until the end of cohabitation. The vaginal smears were stained with Wright's stain before being evaluated. Estrous cycles were not evaluated on the day positive vaginal smears for either sperm or plugs were present.

Postmortem examinations (parental animals):

Complete gross postmortem examinations were conducted on all animals in the study. Selected organs including 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 teh 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 5um and stained with hematoxylin and eosin. Five sections of each ovary from females in the control and high dose were examined for oocyte evaluation.

Statistics:

Quantitative continuous variables (e.g. body weights, food consumption, organ weights, and relative organ weights) were analyzed for statistical significance byBartlett’s test, for homogeneity of variance was used to determine if the groups had equivalent variances at the 1% level of significance. If the variances were equivalent, the groups were compared using a standard one-way analysis of variance (ANOVA). If significant differences among the means were indicated, Dunnett’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, a test for difference in means (Dunn’s Rank Sum test, nonparametric) and a test for ordered response (Jonckheere’s test, nonparametric) were used to determine which treatment groups differed significantly from control.

Pup weight data were analyzed separately by sex using a mixed model analysis of covariance with pups nested within dams, dams nested within dose, and total litter size as the covariate. If differences between groups were identified, the least squares means were calculated to determine which groups differed from the control group. Parental reproductive and offspring survival incidence data were evaluated by χ2 analysis. When the χ2 test indicated group differences, each treatment group was compared to the control group using a 2 × 2 Fisher Exact Test. Armitage’s test for linear trend in the dose groups was also performed.

Sperm and oocyte count data were transformed by Blom’s transformation.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no substance-related, or dose-dependent clinical signs of toxicity in either the P1males or females in any dose group.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male and female body weights in the 0.8% dose groups were significantly (9 to 10%) below control values during the premating period, as werefemale body weights during gestation and lactation

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no substance-related changes in mean food consumption between the treated
and control males and females during the premating period. There were slight differences in mean food consumption between
treated and control animals. These differences included decreases in mean food
consumption of the high dose males during Week 2, and of the high dose females
during Weeks 1, 2, and 6. These small, infrequent differences (<9%) were not
considered treatment-related.

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):

There were substance-related changes in histopathological parameters. 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, which is not human relevant. 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. However, these effects were not considered biologically significant or adverse. The hepatic change, enlarged hepatocytes with increased cytoplasmic eosinophilia, was consistent with peroxisome proliferation (Boorman et al., 1990) and considered to be a physiological adaptation. The current literature suggests that compounds that cause peroxisome proliferation in rodents have little, if any, effects on human liver (Corton, Crit Rev Toxicol. 2014 Jan;44(1):1-49. doi: 10.3109/10408444.2013.835784).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no substance-related effects on oestrous cyclicity.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no biologically significant differences between the control and treated parental animals for reproductive indices, gonadal function parameters, or pathological changes in reproductive tissue. The variation infindings observed were considered spontaneous and were nonadverse.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

> 0.8 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive NOAEL is the highest dose tested = 0.8% Lowest estimated dose for 0.8% = 427mg/kg/bw/d (P1 males, premating) Highest estimted dose for 0.8% = 1,424 mg/kg/bw/d (P2 females, post partum)

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs judged to be directly related to treatment with the test material.
The majority of animals in all groups had no adverse clinical signs during the
premating/mating, postmating, gestation, and/or postpartum periods. There was a very low
occurrence of observations in the male and/or female animals and included dental
abnormalities, scabs/sores, truncated tail, alopecia, ocular/nasal discharge, dry rales,
emaciation, little sign of food consumption/stool, red material around vagina/penis, anogenital
staining, and/or swollen snout/palate. All observations were considered incidental and
unrelated to treatment with the test material.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related deaths. One control male was sacrificed for humane reasons
on Day 43 due to a broken snout. One mid dose female was sacrificed in extremely poor
condition on Day 97 after being observed with little sign of food consumption/stool,
anogenital staining, and emaciation. Postmortem examination of the mid dose female revealed
red material around vagina, moderate emaciation, and small liver and spleen. While the exact
cause of morbidity could not be determined, this death was considered incidental and unrelated
to treatment of the test material.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were substrance-related decreases in mean P1 male/female body weight in the 0.8% dose group.
There were treatment-related statistically significant lower mean body weights in the
high dose males compared with controls at all intervals during the premating period.
Initially, these significantly lower mean body weights were most likely due to the
lower mean body weight of the Fl pups during weaning which remained consistently
lower and were evident at the beginning of the P2 generation. However, as the
premating period continued the difference between the control group and the high dose
group grew larger. At Day 0 of the P2 premating period, the mean body weight of the
high dose males was 7% lower than controls. However, by Day 56 of premating
period, the high dose mean body weight was 14% lower than the controls. From Day
56 until the beginning of sacrifice, the high dose males remained between 13% and
14% lower than controls. Similarly, suppression of body weight gain was observed in
the high dose males compared witii controls throughout most of the study (statistical
significance occurred at intervals 0/7,7/14,21/28,35/42, and 49/56).
In the mid dose males, mean body weights were statistically significantly lower than
the control on Days 56, 70, 77, 84, 91, 98, and 105. However, because these
differences were small (<6%), they were not considered biologically significant.
Additionally, there were several sporadic statistically significant decreases in mean
body weight gain in both the mid and low dose groups compared with controls.
However, in the absence of a clear consistent pattern of response, or significant
differences in the absolute body weight in the low and mid dose groups, these changes
in body weight gain were not considered biologically important.
There were statistically significant higher mean body weights in the low (Days 0 and 7)
and mid dose females (Days 0, 14, and 21) during the premating interval. An increase
in body weight usually is not considered a sign of toxicity and these higher body
weights were not considered biologically significant.
During gestation, there were no statistically significant differences in mean body
weight or mean body weight gain between treated and control females.

During the postpartum period, there were statistically significant reductions in mean
body weights and mean body weight change for the high dose females compared with
controls on PPD 10, 14 and PPD 10/14, respectively. However, this decrease was
followed by a statistically significant increase in mean body weight change for the PPD
14/21 interval for the high dose females compared with controls. There were no
differences in body weight gain between the high dose females and controls for the
overall postpartum interval (PPD 0-21). However, reduced maternal food consumption
and reduced offspring body weight were observed postpartum. Thus,
although not consistently observed, lower mean body weights (7.5-9.3%) during the
postpartum period were considered biologically significant and treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the premating period, statistically significant lower mean food consumption
values were noted in the high dose males compared with controls during the majority
of intervals (up to 11 %). The lower food consumption values correlated with the lower
body weights seen in the high dose males during the premating period.
In the females during the premating period, there were no statistically significant
differences in mean food consumption between treated and control animals, with the
exception of a small (8%) increase in the mid dose females during Week 2. This
increase was considered incidental and not related to treatment with the test material.
During gestation, there were no statistically significant differences in mean food
consumption between treated and control animals, with the exception of a small (11%)
increase in the low dose females during the GD 7/14 interval compared with controls.
This increase was considered incidental and not related to treatment with the test
material.
During the postpartum period, statistically significant lower mean food consumption
was observed in the high dose females compared with controls during the PPD 7/10,
10/14, and 14/21 intervals (16%, 18%, and 17%, respectively), as well as the overall
postpartum period (17%). These lower food consumption values correlated at least in
part with body weight findings and were considered treatment-related.

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, non-treatment-related

Description (incidence and severity):

There were no substance-related changes in male/female organ weights.

There were statistically significant increases in the mean absolute and relative liver
weights of the high dose males (12% and 29%, respectively), high dose females (22%
and 28%, respectively), mid dose females (18% and 13%, respectively), low dose
females (17% and 11%, respectively), and mid dose males for mean relative liver
weight only (18%) compared with controls. These increases were consistent with the
observed histopathology (see page 62) and the known capability of certain phthalates to
cause peroxisome proliferation (Boorman et al., 1990) and are considered to be a
physiological adaptation.

In the kidneys, there were statistically significant increases in mean absolute and relative
weights of the high dose males (19% and 37%, respectively), the mid dose males (18% and
28%, respectively), and the low dose males (9% and 12% respectively) compared with
controls. Microscopic examination of the kidneys revealed changes in all treated male
groups (see page 62),
Differences in kidney weights were also observed in the females and included a statistically
significant increase in mean relative kidney weight for the high dose females (14%), mean
absolute and relative kidney weight of the mid dose females (16% and 11%, respectively),
and mean absolute kidney weight of the low dose females (12%) compared with controls.
However, in the absence of correlating histopathology, these changes were not considered
biologically important.

There were no statistically significant differences in absolute reproductive organ weights
between treated and control animals of either sex except for a decrease in right ovary weight
at 0.8% and the left ovary at 0.4%; and an increase in mean absolute uterine weight at 0.4%.
There were however, statistically significant differences in several mean relative
reproductive organ weights including increases in the mean relative right epididymis
(cauda) weight, total epididymis weight, and seminal vesicle weights of the mid and high
dose males; the mean relative left and right testis weights of the high dose males; and the
mean relative uterine weight of the mid dose females. In the absence of correlating
histopathology, correlating changes in sperm motility, morphology or cauda epididymal
total, and/or a clear consistent pattern of response in both the left and right organs of a
paired set, these differences were considered incidental and unrelated to treatment.

There also was a statistically significant decrease in the mean absolute brain weight (4%) of
the high dose males compared with controls. In contrast, the mean relative brain weight of
the high dose males was increased significantly (p 0.01) compared with controls. Based on
these conflicting differences, the small change in brain weight was considered incidental,
unrelated to treatment, and probably secondary to reduced body weight with brain growth
remaining more stable. There also were increases in the mean relative thymus and spleen
weights in the high dose males compared with controls. Based on the lack of observed
microscopic changes, these differences were also considered to be incidental and probably
secondary to reduced body weight. In the females, there was an increase in mean absolute
and relative adrenal weights of the mid dose group compared with controls. However, in
the absence of a clear consistent pattern of response or correlating histopathology, these
changes were not considered biologically important.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Notable postmortem observations in the animals surviving to termination were limited
to an increased incidence of dilated renal pelves in the high dose males compared with
controls. Dilated renal pelves also were observed in the low and mid dose males, but
the incidence generally was similar to controls. Dilatation of the renal pelves was noted
histopathologically. Dilated renal pelves also were noted in two low dose females.
There also was a low incidence of other postmortem observations in the animals
surviving to termination which included small/flaccid testis, large/flaccid cauda
epididymis, small seminal vesicles, abnormal kidney contents, discolored kidneys,
kidney adhered to the adrenal, large discolored renal lymph nodes, discolored lungs,
misshapen heart, cystic ovaries, distended uterus, mass on the uterus, abnormal
stomach/colon/cecum contents, thick or discolored stomach, accessory liver lobe,
discolored thymus or liver, alopecia, staining of the fur, dental abnormalities, swollen
snout, truncated tail, and/or sores. These observations were considered incidental and
unrelated to treatment.

Postmortem observations in the one control male sacrificed for humane reasons on Day
43 due to a broken snout included emaciation, maloccluded incisors, and dry red ocular
discharge. Postmortem examination of the mid dose female sacrificed in extremely
poor condition on Day 97 revealed red material vagina, moderate emaciation, and small
liver and spleen. The exact cause of morbidity could not be determined, but this death
was considered incidental and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were substance-related histopathology changes in females and males. However, these changes were similar to the P0 generation and were considered nonadverse because these findings are not considered relevant to humans.

Microscopic changes occurred in the liver of male and female rats of all treatment
groups in the P2 animals and consisted of either centrilobular or diffuse hepatocellular
hypertrophy. The affected hepatocytes were enlarged with an increased cytoplasmic
eosinophilia. The incidence and severity of this change increased in a dose-related
manner. This type of hepatocellular change, although not diagnostic, is seen with
compounds which cause peroxisome proliferation (Boorman et al., 1990). This hepatic
change is considered to be a physiological adaptation, and therefore, was not
considered biologically significant. Furthermore, the current literature suggests that
compounds that cause peroxisome proliferation in rodents have little, if any, relevance
'to human health (Corton, 2014).

Microscopic changes occurred in the kidneys of all treated males and included
accumulations of dark orange or eosinophilic granular cytoplasmic pigment in the
cortical tubules and cortical tubular degeneration. Additionally, there was an increased
incidence of granular casts in the renal tubules. These changes appeared to correlate
with the increased kidney weights. In the females, only very small kidney weight
changes were observed and there was no correlating histopathology. These results
appear consistent with male rat-specific nephropathy which is believed to be irrelevant
to humans. Various chemicals induce accumulation of alpha2u-globulin (a2UG), a low
molecular-weight protein, in the male rat kidney. This phenomenon has been
designated a2UG nephropathy (EPA, 1991). a2UG is synthesized in male rats only
under the control of testosterone. Female rats and other laboratory mammals
administered the same chemicals do not accumulate a2UG in the kidney and do not
develop the subsequent a2UG nephropathy.

There was an increased incidence of atrophy of the thymus of the high dose females
compared with controls. This was considered a secondary and stress-related
phenomenon and judged to be a reflection of the lower terminal body weight. Thymic
atrophy was observed in a few female rats of the low and mid dose group, but was not
considered to be treatment-related, as a similar atrophy was observed in a few control
females.
There were no treatment-related microscopic changes in the reproductive organs of
either sex.

All microscopic changes observed in the P2 animals were considered to have
occurred spontaneously, were typical of those which would be expected to occur in rats
of this age, and were considered unrelated to treatment with the test material.

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were substance-related changes in mean body weight and food consumption in 0.8% treated animals. There were no other adverse substance-related findings.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no substance-related effects on oestrous cyclicity at any dose level.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no substance-related or statistically significant differences in mean homogenization resistant
spermatid counts, cauda weight, total cauda sperm count, progressive sperm motility,
or percent normal sperm between the treated and control males.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no substance-related changes in reproductive performance. Although there were statistically significant increases in Male Mating, Male Fertility, and
Female Fertility Indices in the high dose compared with controls, the increases in these
indices are not considered a sign of toxicity and therefore, were not considered
biologically important. There were no statistically significant differences in Female
Fecundity or Female Gestational Indices between treated and control animals. Mean
days of gestation and mean litter size of the treated and control groups were essentially
equivalent. Eleven control, nine low dose, ten mid dose, and two high dose females
were not pregnant.
There were statistically significant differences in the mean sex ratios of the low dose
offspring compared with controls; however, this was not observed in a clear dose-related response manner. Hence, these
differences were considered incidental and nonadverse.

Details on results (P1)

Overall, this study demonstrated little parental toxicity, primarily decreases in body weight and increases in kidney and liver weight with corresponding histologic changes. These results are consistent with previous studies, as well as the ability of phthalates in general to induce peroxisome proliferation. No overt changes in fertility or reproductive function were observed. NOAELs for fertility and reproductive development were approximately 600 mg/kg/day (0.8%) based on the two generation study.

Effect levels (P1)

1

Key result

Dose descriptor:

LOAEL

Effect level:

0.8 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

LOAEL: 0.8% highest dose tested for body weight and food consumption

2

Key result

Dose descriptor:

NOAEL

Effect level:

0.8 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL

Remarks on result:

other:

Remarks:

NOAEL: 0.8% highest dose tested based on reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no substance-related clinical, or postmortem findings. The variation in findings observed were similar to that seen in control animals and was not considered adverse.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In the F1 generation, there was a substance-related decrease in pup survival in the 0.8% dose group on PND 4, but was not reduced at later time points. This decrease was due to 15 out of 43 litters with cannibalized and/or found dead pups. Only 6 litters contained pups without milk in their stomachs. In the 0, 0.2%, 0.4% and 0.8% dose groups, there were 12, 8, 3 and 17 pups (GD 22 to PND 12), respectively from found without milk in the stomach; hence, palatability was not the primary explanation for decrease in pup survival. The maternal behavior of cannibalism-which is a common behavior in rodents to cull their litters or when dams are nutrient deficient- was a contributing factor to the decrease in live birth % is due . Hence, the decrease in pup survival was not due to developmental toxicity, rather than maternal behavior. Additional evidence of bones found in the dams’ stomachs at gross examination supports this. Therefore, the pup deaths were not considered adverse for developmental toxicity, but rather resulting from maternal toxicity. Survival in other dose groups was similar to controls. 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.

Dose (%) Live Birth(%) Day 1 Survival(%) Day 4Survival(%) Day 7Survival(%) Day 14Survival(%) Day 21Survival(%) Viability atWeaning(%)
0 98.7 95.5 93.9 97.8 95.5 100 93.4
0.2 97.6 95.8 93 100 100** 100 98.9
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)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were substance-related decreases in F1 mean pup body weight. Offspring female and male body weights were reduced in the 0.8% dose group at PND 0 (16% and 19% below control, respectively) and these values were statistically significant and outside the laboratory historical control. However, by PND 35, the offspring body weights were similar to control values at 0.8%. Therefore, the changes in body weight at 0.8% were not considered adverse as they were able to recover. There were no changes in body weight in the remaining dose groups.

In the satellite studies, F1off-spring born to 0.8% dose group dams and cross-fostered tocontrol dams on PND 0 had body weights that were similarto those of the main study control offspring throughout thepostnatal phase (data not shown). By PND 14, the meanbody weights of the offspring cross-fostered to the 0.8%dose group dams were significantly reduced (up to 19%) incomparison to the main study control offspring on PND 14and 21. Following weaning, these animals remained oncontrol or 0.8% diets corresponding with their cross-foster-ing treatment, for the second generation premating phase.The mean body weights of the offspring cross-fostered to0.8% dose group dams continued to be statistically signifi-cantly lower (7 to 11% difference) than the mean bodyweights of the offspring cross-fostered to control dams.In switched diet animals, the 0.8% dose group offspringswitched to control diet displayed signs of recovery in bodyweight immediately after weaning and showed normal growth patterns. The control offspring of both sexes switched to the 0.8% dose diet displayed a slight reduction in body weight gain as the study progressed, similar to the main study 0.8% dose group animals during the P1premating inter-val.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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):

There were no substance-related differences in F1 offspring developmental landmarks, anogenital distance,nipple retention, preputial separation, or vaginal patency. There was a statistically significant increase in age at vaginal patency in the 0.4 and 0.8% dose groups (34.2 compared to 32.2 days in control), but overall the difference was minor (#2 days) and within the range of controls. Therefore, these variations were not considered adverse. The remaining dose groups were similar to control.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no substance-related differences in F1 offspring anogenital distance. The variations seen were similar to controls.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no substance-related differences in F1 offspring in nipple retention. The variations seen were similar to controls.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were substance-related findings in organ weights in the F1 generation. Adult organ weight data were similar to those noted in the P1generation. There were significant increases infemale liver weights in all dose groups and in high dose group males. Kidney weights were significantly elevated in malesfrom all dose groups. Left ovary weights were reduced infemales in the 0.8% dose group; however, the uterus weightswere not significantly different when expressed as a fraction ofbody 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. Hence, these findings were not considered relevant to humans; and therefore, were not considered adverse.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no substance -related gross pathological findings. All findings were similar to those seen in control animals.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no pathologic changes in sexual organs from either males orfemales. Similar to the P1animals, histopathologic alterationswere noted in kidneys of treated males and in livers of treated animals of both sexes, but there were no histologic changes in other organs. Based on evidejce that these findings are not relevant to humans, the histtopathologic alterations were not considered adverse.

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.

Remarks:

600 mkd estimated calculation based on 0.8% diet

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: estimated 0.8% is 600 mg/kg/day

Remarks:

NOAEL

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no substance-related effect on clinical signs. T

here were no treatment-related clinical signs observed in the offspring of any group
and the majority of offspring in all groups were free of observable abnormalities from
PND 0-21. Some offspring from all groups were observed without milk in their
stomachs, primarily during the first week of the postnatal period, with the highest
incidence occurring on PND 0.
Single or low incidences of lacerations, sores, scabs, necrotic or truncated tail,
hypothermia, emaciation, ocular opacity and/or swollen umbilicus were observed in
one or more groups, including controls. These observations were considered incidental
and unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was a substance-related decrease in pup survival in the F2 generation. In the F2 generation, there was a substance-related decrease in pup survival in the 0.8% dose group on PND 4, but was not reduced at later time points. This decrease was due to 18 out of 28 litters with cannibalized pups. The calculated % pup survival was 77.6%, compared to historical control range 92.8 % to 99.7% on PND 4. However, when % pup survival was calculated w/ cannibalized pups excluded, the % pup survival was 86.3%. Although 17 litters contained pups without milk in their stomachs between PND 0 to 4, the litters with cannibalized pups did not always correlate with litters with pups without milk in their stomachs. Therefore, palatability does not appear to be the primary contributor to decreased pup survival. Rather, since most pups without milk in their stomachs were found on PND 0, directly after parturition, the evidence for altered dam behavior (i.e., increased pup cannibalism, gross findings of reddened lining of stomach and content of bones and bedding in stomach, neglecting pups, and litter culling) supports early-life stress that transiently alters maternal behavior, including dam-pup interactions, which ultimately led to decreased pup survival. Together, the constellation of endpoints do not suggest developmental toxicity, rather than a treatment-related effect on maternal behavior.

Survival in ten F2 litters (4 low, 2 mid, 4 high dose) was significantly
outside the 99% confidence intervals established for the laboratory's historical control
intervals ( 2.58 standard deviations). This high mortality is inconsistent with the
treatment-related effects observed in this study, or this laboratory's historical control
database. Usually, treatment-related effects on survival involve fewer number of pups
per litter, but across more litters. In addition, it is noteworthy that the Survival Indices
of the F2 controls was often at the low end of the historical control range for this
laboratory.

The Live Birth Index of the low dose group was reduced compared to control.
However this reduction was not observed in the higher dose groups, i.e. there was no
dose response. Thus, this small decrease (4%) was not considered treatment-related.
There were statistically significant decreases in the Day 1 and Day 4 survival indices in
the high dose (12% and 17%, respectively), mid dose (7% and 8%, respectively), and
low dose (5% and 9%, respectively) compared with controls. As addressed in the
discussion, the differences in offspring survival were considered to be the result of
biological variability.

The Day 7 Survival and Viability at Weaning Indices of the high dose offspring were
significantly reduced compared with controls. However, these small decreases (<6%)
were considered to be the result of biological variability. In addition, these values were
within the historical control range of this laboratory and therefore not considered
biologically significant.

Dose (%) Live Birth(%) Day 1 Survival(%) Day 4Survival(%) Day 7Survival(%) Day 14Survival(%) Day 21Survival(%) Viability at Weaning(%)
0 98.5 96.6 94 99.3 99.3 100 98.7
0.2 94.7* 92.1* 85.8 100 100 100 100
0.4 98.2* 89.6* 86.7 99.3 98.5 100 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 92.8–100 93.7–100 98.8–100 86.9–100
*Mean significantly different from controls (P,0.05)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were statistically significant lower mean body weights in the high dose males on
PND 0 and in the high dose males and females on PND 1, 4, 7, 14, and 21. 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.
Additionally, all mean weights of both the male and female high dose offspring at all
intervals were lower than the historical control range of this laboratory. The lower
body weights in the high dose offspring were considered a treatment-related effect.
The lower offspring body weights in the high dose group during the postnatal period
may have been related to maternal stress, changes in the quality and quantity of milk,
decreased milk consumption, and or possibly direct toxic actions of the test material.

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

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were statistically significant decreases in the mean absolute brain and kidney
weights of the high dose males and females and corresponding increases in mean
relative brain and kidney weights of the high dose males and females compared with
controls. Additionally, in the mid dose group, there was a statistically significant
decrease in mean absolute brain weights in females and an increase in mean relative
kidney weights for both sexes compared with controls. However, microscopic
examination of the brains and kidneys revealed no microscopic changes. Therefore,
these differences in the kidneys were probably an adaptive change without biological
significance and the differences in the brain were probably the result of decreased body
weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

there were no gross postmortem observations in the F2 offspring judged to
be related to treatment with the test material. The majority of animals were free of
observable abnormalities at the scheduled terminal sacrifice on PND 21. Observations
were limited to single occurrences of discolored stomach, discolored thymus, adrenal
adhered to kidney, ocular opacity, and a low incidence of dilated renal pelvis. These
limited observations were considered incidental and unrelated to treatment.
Four high dose male offspring were noted with undescended testes. This effect was
probably related to slow development in the high dose males as indicated by decreased
body weight during the entire postnatal period.

The majority of animals which died prior to scheduled termination (PND 0-20) also
were free of observable abnormalities. There were single incidences of anasarca (low
dose), distended gastrointestinal tract (low dose), thick/enlarged atria (low dose), small
pale heart ventricle (control), dilated renal pelvis (control and low dose), and truncated
tail (control and mid dose). Due to their isolated incidence, all postmortem
observations were considered incidental and unrelated to treatment with the test
material. There was also a low incidence of offspring with scabs/lacerations and no
milk in stomach in all groups including controls, which were considered incidental and
unrelated to treatment with the test material.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment-relatedmicroscopic changes were seen in the liver of the mid and high dose
offspring of both sexes and consisted of an enlargement of the hepatocytes with an
increase of cytoplasmic eosinophilia. The incidence of this change occurred in a dose-related
manner. As in the parental animals, this hepatic change is seen with
compounds which cause peroxisome proliferation (Boorman et al., 1990). This hepatic
change is considered to be a physiological adaptation, and therefore, was not
considered biologically significant. Furthermore, the current literature suggests that
compounds that cause peroxisome proliferation in rodents have little, if any, relevance
'to human health (Corton, 2014). There were no treatment-related microscopic changes observed in the kidneys and
brain.

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

In the high dose, offspring survival was affected on PND 1 to 4, and more in the F2 than the F1 generations. The decrease in pup survival was considered as a developmental toxicity endpoint. However,when individual pup data was examined, pup death was due to an increased incidence of the maternal behavior:cannibalism, and this is not considered a developmental toxicity endpoint. Hence, the NOAEL is based on pup body weight at 0.2%.

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

> 0.2 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: for decreased pup survial, when cannibalized pups were excluded from the % pup survival calculation, % pup survival was within historical control values

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

0.8 other: %

Any other information on results incl. tables

Parental toxicity: In the P1 generation, there were statistically significant increases in the mean absolute and relative liver weights of the 0.4% dose males 12% and 14%, respectively) and 0.4% dose females (12% and 13%,respectively)  compared with controls.  These increases were consistent with findings in the previously conducted two-generation reproductive study (Exxon Biomedical Sciences, 1997d), and related to the known capability of DIDP to cause peroxisome proliferation.  There were statistically significant increases in mean absolute and relative kidney weights of the 0.4% males (14% and 18%, respectively).  These increases were also consistent with findings in the previously conducted two-generation reproductive study (ExxonBiomedical Sciences, 1997d).  There also was a statistically significant increase in the 0.4% female mean relative kidney weight (6%) compared with the controls.  In the P2 generation, there were statistically significant increases in the mean absolute and relative liver weights of the 0.4% males(13% and 14%,respectively), 0.4% females (23% and 20%, respectively), and 0.2% females (17% and 9%, respectively)compared with controls. In the kidneys, there were statistically significant increases in mean absolute and relative weights of the 0.4% dose males (20% and 19%, respectively), and the 0.2% males (10% and 7%,respectively) compared with controls.  There was a statistically significant increase in mean absolute kidney weight in the 0.2% females (13%) compared with controls.  There were no treatment-related deaths.  There were no gross postmortem observations judged to be related directly to treatment with the test material.  The majority of P1 and P2 animals throughout the groups were free of observable abnormalities at postmortem examination.  In the females, there was an apparent dose-related increase in thick and/or discolored stomachs.  This stomach irritation was attributed to ingestion of bedding materials since it was observed only in females and observed in all groups, including controls.  Notable postmortem observations in the P2 animals surviving to termination were limited to an increased incidence (8/29) of dilated renal pelves in the 0.4% dose males compared with controls.  Dilated renal pelves also were observed in the other treated groups (2-5/30),but the incidence generally was similar to controls (3/30).  Dilated renal pelves also were noted in several females.  There were no statistically significant differences in the mean body weight between the treated and control males or females during the P1 or P2 generation, including the gestation and postpartum intervals.

 

Offspring toxicity: There were no biologically significant differences in F1 survivorship between the treated and control offspring and all survival indices were within the historical control range for this laboratory.  Statistically significant differences were limited to an increase in the live birth index of the 0.06% and 0.4% dose groups compared with controls.  These increases were not considered biologically important. In the F2 generation, there was a dose-related decrease in the Day 1 and Day 4 survival indices, with statistically significant decreases being observed in the 0.2% dose group (4% and 10%, respectively) and0.4% dose group (6% and 13%, respectively) compared with controls.  These values were outside the historical control range of this laboratory and were considered treatment related.  These results were consistent with the decreased F2 survivorship in the previous two-generation study.  There were no statistically significant differences between the control and treated animals for post-implantation loss.  The live birth index for the 0.2% dose group was higher than the historical control and this was not considered biologically important.  There were statistically significant increases in Day 14 and viability at weaning indices of the 0.02% and 0.06% dose groups compared with controls. These increases were not considered biologically important.  The Day 21 survival indices of the 0.2% and 0.4% dose groups were marginally outside the historical control range for this laboratory, but not statistically significantly different from the control.  No biological importance was assigned to these observations (cf. Table 4.31).  In the F1 offspring, there were no statistically significant differences in mean body weights between treated and control animals of either sex up to PND 21 nor statistically significant differences in mean body weight or mean food consumption between treated and control offspring of either sex during the two-week post weaning measurements.  In the F2offspring, there were statistically significant lower mean body weights in the 0.4% males on PND 14, the 0.4%females on PND 14 and 21 and the 0.2% females on PND 14 compared with controls.  Although, these weights were within the historical control range of the laboratory, these may have been a treatment-related effect.  There also was a statistically significant increase in the 0.02% male mean body weight on PND 21.  This increase was not considered biologically important. Mean post weaning body weights were significantly decreased compared to controls in the 0.4% dose males during PNDs 28 and 35, and in the 0.2% dose males at PND35 only.  At PNDs 42, 49, and 56, an apparent recovery occurred in the 0.2% and 0.4% treated males and their mean body weights were no longer statistically different from controls.  There were no statistically significant differences in post weaning body weights between treated and control females on PND28.  There were no treatment-related clinical signs observed in the F1 or F2 offspring of any group and the majority of offspring in all groups were free of observable abnormalities from PND 0-21 and during the post weaning periods.  However, there was an increased incidence of cannibalization of F2 pups at PND 1 or 2,not sporadically but a few P2 females in the 0.2 and 0.4% groups were specifically concerned (for instance in the 0.4% treated group one female cannibalized all its litter, e.g. 10 pups/10).  In general, there were no gross postmortem observations in the F1 or F2 offspring judged to be related to treatment with the test material.  The majority of animals selected for necropsy were free of observable abnormalities at the scheduled terminal sacrifice on PND 21.  The majority of animals that died prior to weaning (GD 22 - PND 21) also were free of observable abnormalities.  In the F1 generation, there were no statistically significant differences in mean absolute or relative organ weights (kidney or liver) between treated and control animals of either sex.  In the F2 generation, there were no statistically significant differences in mean absolute or relative organ weights(kidney or liver) between treated and control animals of either sex with the exception of the 0.4% dose group female mean relative liver weight.  There was a statistically significant increase in the mean relative liver weight of the 0.4% dose group females compared with controls.  In the absence of a similar trend in the respective absolute liver weight, this single difference was considered the result of the lower mean bodyweights of the 0.4% females at study termination and not treatment-related.  There were no statistically significant differences in F1 or F2 offspring mean PND 0 anogenital distance between treated and control animals of either sex.  Nipple retention was similar between treated and control offspring of both sexes: the majority of females in all groups had six nipples retained on PND 13/14, while all males in all groups had zero.  In the F1 animals, there were no statistically significant differences in age or weight at preputial separation between treated and control male offspring.  There were no statistically significant differences in age or weight at vaginal patency between treated and control female offspring.  In the F2 animals, there was a statistically significant delay in preputial separation for the 0.4% males when compared to the control male offspring.  This delay was small (1.2 days) and preputial separation was still included within the historical data of CD-rats(Bates et al., in Developmental Toxicology Handbook, 1997).  There were no statistically significant differences in the mean body weight at which preputial separation occurred between treated and control male offspring.  There were no statistically significant differences for age of vaginal patency between treated and control female offspring.  However, there was a statistically significant decrease in the mean body weight at the time that the 0.4% females achieved vaginal patency compared with the control female offspring.  This decrease was small (6%) and not considered biologically significant.

 

Satellite studies: Cross fostering: In the cross fostering satellite study, offspring born to high-dose dams and cross-fostered to control dams on PND 0 exhibited body weights which were not different from main study control offspring throughout the postnatal phase. Conversely, the mean body weights of the offspring cross-fostered to the high-dose dams were statistically significant lower (up to 19%) than the main study control offspring of both sexes on PND 14 and 21. This indicates that DIDP may be transferred through the milk but at a low level, evidenced by a low decrease of body weight; a statistical level of significance was obtained when lactation exposure effects and direct toxicity via feed (solid food is absorbed by pups from PND 14) were combined. Following weaning, these animals remained on control or high-dose diet corresponding with their cross fostering treatment, for the second generation premating phase. The mean body weight of the offspring cross-fostered with high-dose dams continued to be statistically significant lower (9-11% males; 7-10% females) than the mean body weight of the offspring cross-fostered with control dams during premating. In parent equivalents (adult rats stemming from cross-fostered pups), during the premating period there were statistically significant increases in the mean absolute and relative kidney weights of the pups cross fostered with high-dose dams (an increase of respectively 16% and 30% in males, and respectively 9% and 22% in females) compared with pups cross-fostered with control dams. The same trend was observed for liver weights: the mean absolute and relative liver weight of the cross-fostered high-dose group was increased compared with the cross-fostered control group (an increase of respectively 11% and 23% in males and 22% and 35% in females). Pertaining to reproductive organ weight changes in males, mean absolute right and left testis weight of the cross-fostered high-dose group were statistically significantly decreased compared with the cross-fostered control group. Since, relative right and left testis and epididymis weights were increased, this effects is probably due to lower body weight. In females there was an increase of the uterus, right and left ovary weights, only statistically significant when expressed relative to body weights. In absence of histopathology, it could not be determined if changes in tissue structure and function occurred.

 

Switched diet phase:  In the switched diet phase, weaning from high-dose animals was given control diet, while weaning from control animals was given high-dose diet. The high-dose offspring of both sexes switched to control diet displayed signs of recovery in body weight immediately after weaning and displayed normal growth patterns. However a trend toward lower body weight similar to the main study high-dose males was observed after day 42. The control offspring of both sexes switched to high-dose diet displayed slight reduction of body weight gain as the study progressed, similar to the main study high-dose animals during the P1 premating interval. In addition, in the switched diet high-dose P2 equivalents (adult rats stemming from the switched diet pups) there were statistically significant increases of the absolute and relative liver weights (an increase of respectively 36% and 34% in males, 31 and 39% in females) and kidney weights (an increase of 27% in males, respectively 15% and 23% in females), right and left testis and epididymis weights compared with the switched diet control P2 equivalents. The increase of testicular weight observed in males might be related to transient hypothyroidism in early phases of development. Results from the cross-fostering and switched diet satellite groups indicate that lactation exposure may participate to toxicity of DIDP (EU RAR on DIDP (2006)).

 

Summary: No-Observed-Adverse-Effect Levels (NOAELs) for fertility was 0.8%, and for offspring survival was 0.06% (F2 gen, PND 1 -4).  There were no important or dose-dependent clinical signs of toxicity in either the P1 males or females in any dose group.  There were no treatment-related deaths or gross postmortem observations judged to have been related directly to treatment with the test material in any P1 or F1 animals. Increased liver and kidney weights were found at all dose levels in male and female adults.  There were no differences observed in mating, male or female fertility, gestational index, or length of gestation.  Furthermore, there were no differences in mean littersize or sex ratio.  The percentage of live offspring was significantly decreased in the 0.8% dose group and below the laboratory historical control range (HCR).  In addition, reduced offspring survival was observed at postnatal days 1 and 4 in the F2 generation (but not in the F1 generation) at levels of 0.2% DIDP and greater.  There was a statistically significant increase in age of vaginal patency in the 0.4 and 0.8% dose groups, but overall the differences were small (approximately 2 days) and with in the range of unknown biologic relevance.  No clinical signs of toxicity or gross postmortem observations were noted in the offspring. There were no differences in viability at weaning during the postnatal period.  There were no differences in F1 offspring developmental landmarks, anogenital distance, nipple retention, preputial separation, or vaginal patency.  There were no differences in testicular weights in either parents (P1) or offspring (F1) and there were no pathological changes in the testes.  Further, there were no differences in offspring body weight during the postnatal period to PND 35. Results from the cross-fostering and switched diet satellite groups indicate that lactation exposure may participate to toxicity of DIDP (EU RAR on DIDP (2006)).

Applicant's summary and conclusion

Conclusions:

There were no reproductive toxicity findings in females and males in the P1 and P2 generations. Decreased male and female mean body weight gain in the 0.4% and 0.8% dose groups was observed. Therefore, the NOAEL for reproductive toxicity was 0.8% and developmental toxicity was 0.2%.

Executive summary:

There were no statistically significant differences in male mating, male fertility, female fertility, female fecundity, or female gestational indices between treated and control animals in the P1 or P2 generation. Mean days of gestation and mean litter size and of the treated and control groups were similar. There were no statistically significant differences in the mean sex ratio of the treated offspring compared with controls.

Parental toxicity: 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.  In the first study, minor effects on the liver were observed at 0.2% (103 – 203 mg/kg/day).  In the second study, no hepatic effects were recorded at this concentration (114 – 225 mg/kg/day).  As there is a range in intake levels, it is likely this dietary concentration results in ingestion of DIDP at or near the NOAEL for systemic effects from repeated doing.  Up to the highest dose tested no overt signs of reproductive toxicity were reported and no effect was observed on fertility parameters.

 

For offspring toxicity, a decrease in survival indices (day 1 and day 4) in F2 generation was due to maternal behavior (i.e., cannibalism and/or pup neglect due to stress), which is not considered developmental toxicity.  For example, when cannibalized pups were removed from the %pup survival calculation, the % pup survival was slightly outside historical control values (see above endpoint parameter discussion). Therefore, the decrease in pup survival was due to a maternal behavior resulting from maternal toxicity and should not be classified as developmental toxicity. No effect was observed on developmental landmarks assessed at any dose tested; however there was a decrease in mean pup body weight that was considered test article-related at 0.4% and 0.8%. Hence, the NOAEL for offspring was 0.2%.