Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a 2-generation study (OECD 416) in Sprague-Dawley rats, S-216A fed in the diet at up to 5000ppm (ca. 375mg/kg bw/d in males, 333mg/kg bw/d in females) was without effect on any fertility or developmental parameter. Minor effects on sperm morphlogy alone were within the limits of the historical control database and are considered not to be toxicologically relevant.

In a modified combined repeated dose toxicity study with the reproduction/developmental toxicity screening test inSprague-Dawley rats (OECD 422), S-216A fed in the diet at up to 6000ppm (ca. 299 mg/kg bw/d in males, 367 mg/kg bw/d in females) was without effect on any fertility or developmental parameters.


In an extended one-generation reproductive and developmental toxicity study, groups of female Sprague-Dawley rats (25/dose) were exposed to Santicizer 261A at concentrations in the diet of 0, 750, 3750 or 7500 ppm from GD 6, through lactation, to weaning on PD 21. Reduced body-weight gain was seen in the F1 rats during the lactational period in all treatment groups. A NOAEL of 750 ppm (about 50 mg/kg bw/day) was determined for maternal toxicity and also F1 male developmental effects, whereas the data indicate a NOAEL of 750 ppm (about 50 mg/kg bw/day) for maternal reproductive toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Deviations did not compromise the outcome of the study
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC (males and females to be obtained from separate breeding rooms to ensure brothers and sisters were not used)
- Age at study initiation: 5 to 6 weeks upon arrival
- Age at start of exposure: 6 to 7 weeks
- Weight at study initiation: Males: 151-200 g, Females: 150-175 g
- Housing: Animals were individually housed upon arrival, during acclimation period, and upon the initiation of the treatment period in solid-bottom polycarbonate cages suspended on automatic watering racks with filter sheets. The cage dimensions were 8"x19"x10.5" (height) for all phases of this study. Study animals were housed 2 per cage (1 male and 1 female from the same dose level) during the mating period. Females were caged individually once they had successfully mated (or at the end of the mating period). Females were housed indivithroughout the lactation period.
- Diet (e.g. ad libitum): Purina Certified Ground Rodent Chow No. 5002 (Purina Mills, Inc., Richmond, IN)
- Water (e.g. ad libitum): Automatic watering system
- Quarantine: Approximately 1 week
- Acclimation period: Six days under test conditions
- Number and Sex: 140 females, 140 males (25/sex/group, 5 groups, 15/sex sentinels)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±3 (74°F ± 5°F)
- Humidity (%): 30-70% RH
- Air changes (per hr): 10-15 per hour
- Photoperiod (hrs dark / hrs light): 12-hour light: 12-hour dark, with the exception of change over to and from daylight savings time.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): A storage stability study of the bulk chemical at 75 and 5000 ppmdietary concentrations was conducted prior the start of the study with 6 time points: 0, 7, 21, 35, 42 and 52 days.
- Mixing appropriate amounts with (Type of food): The chemical was diluited in acetone over a small portion of blank feed. The mixture was stirred under nitrogen until the acetone was evaporated. The formulation was prepared by layering additional blank feed, the premix, and more blank feed in a twin-shell V blender and mixing for approximately 15 minutes.
- Storage temperature of food: Room temperature for 52 days and seven days ambient in glass feed jars with stainless steel lids.
- After confirmation of storage stability, the formulations were prepared approximately every 5 weeks.

VEHICLE
- Justification for use and choice of vehicle (if other than water): route of exposure
- Concentration in vehicle: 5000 ppm
- Lot/batch no. (if required): Purina Certified Ground Rodent Chow No. 5002
- Purity and Analysis: Provided by supplier with comparison to acceptable levels of contaminants
Details on mating procedure:
- M/F ratio per cage: 2 animals (1 male and 1 female) from the same dose level per cage
- Length of cohabitation: 2 weeks
- Proof of pregnancy: All vaginal smears were dried, sprayed with fixative, stained with toluidine blue, and coverslipped for determination of stages of the estrous cycle and estrous cyclicity for the 3-weeks pre-mating period.
Vaginal smears taken during mating period will be examined for presence of sperm.
- After successful mating (or at the end of the mating period) each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose concentration analysis was conducted using the validated GC/FID method along with the finilised and approved analytical method. The analysis was conducted for the first, middle, and last mixes during the study.
Homogeneity of the dosed feed formulations was evaluated using the same batch size as required for the animal study and at the lowest and highest proposed dietary concentrations. Samples for analysis were collected from left, right, and bottom blender ports for each formulation. Dose concentration analysis was performed for each formulation, and homogeneity was performed once during the prestudy chemistry phase at 75 and 5000 ppm dietary concentrations.
Duration of treatment / exposure:
10 weeks prior to mating, during mating (up to 2 weeks), gestation (3 weeks), lactation through to weaning (3 weeks)
F1: possible indirect exposure during mating, gestation, lactation, 2 week holding period between weaning and the start of the 10 week premating phase
Frequency of treatment:
Dosed feed or vehicle was available in the diet ad libitum 7 days/week.
Details on study schedule:
- F1 parental animals not mated until 10-12 weeks after selected from the F1 litters.
Remarks:
Doses / Concentrations:
0, 250, 750, 2500, 5000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The highest concentration (5000 ppm) was chosen to induce parental toxicity or level of lethality (<=10%) and was provided by the Sponsor, with concurrence of the Study Director, based on data from a one-generation reproductive toxicity study previously conducted at RTI. The lowest concentration (250 ppm) was selected to be an anticipated parental/offspring no observable adverse effect (NOAEL). The mid-doses (750 and 2500 ppm) were selected based on the expectation of offspring developmental and systemic toxicity which occurred in this range of doses in the previous one-generation reproductive toxicity study.
- Rationale for animal assignment (if not random): The animals were assigned to groups based on randomization of body weight.
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily for cageside observations, weekly for out-of-cage observations

BODY WEIGHT: Yes
- Time schedule for examinations: once per week
F0 Males and Females: Once prior to randomization
F0 Male prebreed: Weekly beginning on Day 0 for 10 weeks
F0 Female prebreed: Weekly beginning on Day 0 for 10 weeks
F0 Female mating: Weekly until sperm/plug positive or mating is over
F0 Postmating: Weekly until littering occurs or until necropsy
F0 Female Gestation: GD 0, 7, 14, 20
F0 Female Lactation: PND 0, 4, 7, 14, 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Oestrous cyclicity (parental animals):
Vaginal smears and Estrous were observed daily for last 21 days of prebreed period, placed onto labeled, gridded microscope slides. During the mating period, observations were recorded daily until sperm/plug positive.
All vaginal smears were dried, sprayed with fixative stained with toluidine blue, and coverslipped for determination of stages of the estrous cycle and cyclicity for 3-week premating period.
Vaginal smears taken during mating period were examined for presence of sperm.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes. Litters were standardized to 5 males and 5 females; parial adjustment was acceptable if necessary.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: abnormalities, clinical signs, weight, number and sex of pups, stillbirths, live births, postnatal mortality, anogenital distance.

GROSS EXAMINATION OF DEAD PUPS:
yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: during the lactation period once litters had been born
- Maternal animals: All surviving animals: sacrifice of each animal occurred on her PND 21. Sacrifice of animals that never littered occurred prior to PND 21 day.

GROSS NECROPSY
- Gross necropsy: A complete gross necropsy was conducted after death of any parental or offsping due to euthanasia or accidental death. The gross necropsy included examination of the external surfaces; all orifices, carcass, the thoracic abdominal and pelvic cavities and their viscera (including reproductive organs, and cervical tissues and organs.Uteri of females were examined for number of nidation (implantation) scar.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathology examination of the designated organs was performed for all high dose (group 5) and control (Group 0), parental (F0 and F1) males and females.
Additional organs of the low and mid dose animals were included.
The tissues indicated below were prepared for microscopic examination and weighed, respectively:
- Uterus with Oviducts and Cervix
- Ovaries (paired)
- Vagina
- Testes (paired)
- Epididymides (paired)
- Seminal Vesicles With Coagulating Glands and Their Fluids
- Prostate (whole)
- Brain
- Pituitary (weighed fixed)
- Liver
- Kidneys (paired)
- Adrenal Glands (paired)
- Spleen
- Thyroid (weighed fixed)
- Lungs
- Gross Lesions
Postmortem examinations (offspring):
SACRIFICE
- All nonselected weanlings on PND 21 with external abnormalities or clinical signs, as well as at least 1 arbitrarily selected weanling/sex/litter (to yield at least 3 weanlings/sex/litter if possible) in all groups, for both F1 and F2 weanlings, will be terminated (on PND 21) and subjected to a complete gross necropsy after euthanasia.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations: all orifices; carcass; the thoracic, abdominal, and pelvic cavities and their viscera (with special attention paid to reproductive organs); and cervical tissues and organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
- Brain
- Spleen
- Thymus
- Liver
- Gross Lesions
Statistics:
Quantitative continuous data (e.g., adult and pup body weights and weight gains, feed consumption in g/day and g/kg body weight/day) was analyzed using 1-way analysis of variance (ANOVA) or nonparametric analysis of variance, pairwise tests (Dunnett, 1955; 1964) for parametric and nonparametric data, and Levene’s test (Levene, 1960) or Bartlett’s test (Bartlett, 1937) for homogeneity of variance.
When appropriate, analysis of covariance was used. The criterion for statistical significance of comparisons was p<0.05.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
increased % abnormal sperm, within historical control (see Table 1)
Reproductive performance:
no effects observed
F0 Parental Mortality
All F0 males and female (25/group/sex and five groups) survived to scheduled necropsy except one F0 female (250 ppm) found moribund on Lactational Day (LD) 2 one F0 female (2500 ppm) found dead on LD 0.

F0 Males
F0 Male Clinical Observations
No dose-related clinical observations. In one or two males per group, alopecia, eye discharge, rough coat and/or sore(s) on the body were observed.

F0 Male Body Weights/Weight Gains and Feed Consumption
F0 male body weights were equivalent across all dose groups from day 0-7 through day 98-105 as well as F0 male absolute body weight gains from day 0-7 through day 91-98. A significant reduction was recorded at day 56-63 at 250 and 750 ppm only. A significant increase was recorded for males not yet necropsied on day 98 (p<0.05) from day 98-105, at 2500 and 5000 ppm.

F0 male feed consumption values were equivalent across all dose groups from day 0-7 through day 98-105.
Intake values of the test material are:
- 0 ppm: 0 mg/kg/day for all weekly intervals
- 250 ppm: 23-11.5 mg/kg/day for days 0-7 through days 98-105
- 750 ppm: 67.4-34.3 mg/kg/day for days 0-7 through days 98-105
- 2500 ppm: 228.5-112.4 mg/kg/day for days 0-7 through days 98-105
- 5000 ppm: 450.8-224.1 mg/kg/day for days 0-7 through days 98-105

F0 Male Necropsy
Terminal body weights and all absolute and relative organ weights were equivalent across all dose groups except for:
- Kidney: increased relative (but not absolute) right (but not left)
- Liver: significantly increased (at p<0.05) at 5000 ppm, increased absolute weight at 5000 ppm (at p<0.01), and increased relative weight at 2500 and 5000 ppm (both at p<0.001).

Andrology data showed no effects at any dose for any parameter except for a slight but statistically significant increase in the percentage of abnormal sperm at 750 ppm (p<0.05) and at 2500 and 5000 ppm (both at p<0.001). All valuies were wiithin the historical control (Table 1).

Gross pathology showed no findings in the higher dose groups (2500 and 5000 ppm) except, possibly, for misshapen testes, bilateral, observed in one male in the 5000 ppm treatment group.

F0 generation males indicated that administration of Santicizer 261A (S-261A) in the diet to Crl:CD(SD) rats, under the conditions of this study, was not associated with any treatment or dose related male reproductive organ histopathology at any dose.

F0 Females
F0 Female Clinical Observations
No clinical findings have been recorded during the prebreed period at any dose except for alopecia in one-three females at 250,750, and 5000 ppm, one-two females with sore(s) on the body at 250, 2500, and 5000 ppm, eye discharge (one at 2500 ppm and one at 5000 ppm), scab (one at 5000 ppm), and vaginal thread (not a clinical observation per se) in one female each at 2500 and 5000 ppm.

Weekly body weights and body weight changes were equivalent across all dose groups from day 0 through 70.
F0 female feed consumption values in g/day and in g/kg/day were equivalent across groups for any weekly interval or by week within groups.

Intake values are:
- 0 ppm: 0.0(start)-0.0 (end)
- 250 ppm: 22.9-17.0
- 750 ppm: 0.3-53.6
- 2500 ppm: 254.8-186.4
- 5000 ppm: 457.6-338.9 mg/kg/day

Vaginal cytology evaluation showed no differences among groups for numbers of females cycling, estrous cycle length in days, numbers of estrous cycles during the examination period, the incidences of irregular cycles, or of abnormal cycles.

F0 Gestation
Clinical observations during gestation were limited to sore(s) on body, alopecia and/or eye discharge in one-three females/group. No treatment or dose relationship.

Body weights and body weight changes during gestation, on GD 0, 7, 14, and 20, were equivalent across all groups. A significant reduction in gain weight was observed at 750 and 5000 ppm (p<0.05) only for week 1 (GD 0-7) of gestation; a significant reduction in body weight gain (p<0.01) for the entire gestational period, GD 0-20, only for F0 females at 250 ppm.

F0 Female Necropsy
No significant differences were found among groups for F0 female terminal body weight, left or right absolute or relative adrenal weight, brain weight, kidney weight, or lung, pituitary, spleen, or thyroid weights, ovarian weights, uterus with cervix and oviducts.
- Relative right kidney weight: significant increase at 2500 ppm (p<0.01),
- Absolute liver weight: significant increase at 5000 ppm (p<0.05)
- Relative liver weights: at 2500 and 5000 ppm (p<0.05)

No differences across groups for stage of estrus at necropsy: diestrus, estrus, metestrus (most of the females), or proestrus (fewest of the females) were recorded. The estrous stage at necropsy could not be determined on only two females at 750 ppm.

No gross pathology findings were recorded at the higher incidence in the two highest dose groups (2500 and 5000 ppm), except for one clear cyst on the left ovary in one female at 5000 ppm, and one animal with alopecia on the forefoot at 5000 ppm; alopecia on other limb locations was noticed in animals at other doses.
None of these gross findings appeared treatment or dose related.

Histopathologic evaluation indicated a potential treatment-related histopathological changes in the high dose females, including a small but increased incidence of bilateral cortical vacuolation of the adrenal glands. No reproductive histopathology was observed in F0 females at any dose.

F1 Parental Animals
F1 Males and Females
The F1 males and females selected to be parents of the F2 generation survived to scheduled sacrifice. One female was euthanized on Lactational day 0.

F1 Males
There were no treatment/dose-related incidences of clinical observations for F1 adult parental males at any dose, from day 1 to day 106.
Age at acquisition of preputial separation for the F1 males was equivalent across all groups. Body weight at acquisition was significantly decreased (at p<0.01) only at 5000 ppm.

F1 male body weights were significantly reduced only at 5000 ppm at p<0.01 for Days -14 and-7; at 5000 ppm at p<0.001 for Days 0, 7, 14, and 21, and at p<0.05 for Days 35, 42, and 49. Body weights from days 56 through 105 were statistically equivalent across all groups.
Body weight changes were significantly reduced for Day -7 to Day 0 at 5000 ppm (at p<0.05) and for Days 0-7 at 5000 ppm (at p<0.01), and at 2500 ppm for Days 56-63 (at p<0.01) and at 5000 ppm for Days 70-77 (at p<0.05). Body weight changes for all remaining weekly intervals were statistically equivalent across all groups.

Feed consumption values were:
- Significantly reduced: at 5000 ppm and only for Days 0-7 (at p<0.05).
Feed consumption (g/kg/d) was equivalent acrsoss all other groups and time points.
Relative feed consumption was:
- Significantly increased: at 5000 ppm for Days -7 to 0 and for Days 0-7 (at p<0.01) and Days 7-14 (at p<0.001)
- Significantly increased: 250 ppm (at p<0.01) for days -7-0 and at p<0.05 for days 7-14 and at 750 ppm (at p<0.05) for Days 7-14.
Feed consumption in g/kg/day for all remaining weekly intervals was equivalent for all doses.

Intake values:
- 0ppm: 0 for all weekly intervals
- 250 ppm: from 38.6 (Days -14 to -7) to 11.4 mg/kg/day (Days 98-105);
- 750 ppm: from 115.3 mg/kg/day (Days -14 to -7) to 33.1 mg/kg/day (Days 98-105)
- 2500 ppm: from 383.9 (Days -14 to -7) to 113.5 mg/kg/day (Days 98-105)
- 5000 ppm: from 776.8 (Days -14 to -7) to 217.4 mg/kg/day (Days 98-105).

No significant differences were found in the F1 male terminal body weight, left or right absolute or relative adrenal weight, brain weight, kidney weight, or lung, pituitary, spleen, or thyroid weights, ovarian weights, uterus with cervix and oviducts.
- Absolute liver weight: unaffected across all groups.
- Relative liver weight: statistically significantly increased at 750 ppm (at p<0.05) and at 5000 ppm (at p<0.001), and increased, but not statistically significantly, at 2500 ppm.

Andrology data showed no effects at any dose for any parameter except for a slight but statistically significant increase in the percentage of abnormal sperm at all concentrations (p<0.001). All values were within historical control (table 1).

No pathology finding were recorded for the top or doses.
Male histopathology indicated a small, but increased, incidences of bilateral cortical vacuolation that could potentially be related to the test article. None males at 5000 ppm, none (0) at 2500 ppm (adrenal glands were not examined histopathologically at 250 ppm and 750 ppm) and 2 at 0 ppm showed this effect.
No reproductive histopathogy findings in the F1 males at any dose were recorded.

F1 Females
Clinical observations were done from the holding period between the weaning of the first F1 litter to the start of the 10-week prebreed period. No findings were recorded.
The acquisition of vaginal patency (and the body weight at acquisition) was unaffected across all groups.

F1 female body weights and body weight changes indicated no statistically (or biologically) significant differences among groups for any of the weekly body weights.

The feed consumption values for days 0-70 were equivalent at 0, 250, 750 and 5000 ppm (from 20.94 to21.96 g/day), and significantly increased at 2500 ppm (to 24.43 g/day, p<0.01). No differences across groups for feed consumption from day 84-112 for the few remaining females not sperm- or plug-positive.
- Significant increases: at 250 ppm (at p<0.05) and at 2500 ppm (at p<0.001) for days 0-7; at 250 and 2500 ppm (both at p<0.05) for days 28-35, at 2500 ppm (at p<0.01) for days 0-70
- No effects: at 750 or 5000 ppm. Values for day -14 to -7, -7 to 0, and for most weeks during the prebreed period, for days 0-70 inclusive, and for days 84

Intake values:
- 0 at 0 ppm from 39.31 (days -14 to -7)
- 250 pp: to 17.06 mg/kg/day (days 63-70, the last date when all females per group were present)
- 750 ppm: from 122.74 to 49.29 mg/kg/day
- 2500 ppm: from 406.27 to 187.02 mg/kg/day
- 5000 ppm: from 808.69 to 341.50 mg/kg/day at 5000 ppm (same interval)

Vaginal cytology analysis indicated that cycling, mean estrous cycle length was equivalent across all groups (4.10 to 4.29 days) except for the 2500 ppm group with a significantly longer mean estrous cycle length of 4.60 (p<0.05).

F1 female clinical observations were equivalent across all groups. F1 female body weights, body weight changes, feed consumption values during gestation were equivalent across all groups.

Intake values:
- 0 mg/kg/day at 0 ppm for GD 0 to 20,
- 250 ppm: 17.76 to 14.78 mg/kg/day
- 750 ppm: 50.75-44.57 mg/kg/day
- 2500 ppm: from 170.52 to 163.08 mg/kg/day
- 5000 ppm: from 357.02 to 300.83 mg/kg/day

No clinical observations during lactation (LD 0-21) were recorded at the top doses. One female in the control group lost her entire litter by LD 5 and one at 750 ppm lost LD 1. One other female in the control group was euthanised on LD0.
F1 female body weights, body weight changes, feed consumption during lactation indicated no differences among groups for mean body weights on any weigh day.

Intake values:
- 0ppm: 0 mg/kg/day
- 250 ppm: from 22.51 (on LD 0-4) to 49.11 mg/kg/day ( LD 14-21)
- 750 ppm: from 67.68 (LD 0-4) to 145.09 mg/kg/day (LD 14-21)
- 2500 ppm: from 233.95 (LD 0-4) to 493.09 mg/kg/day (LD 14-21)
- 5000 ppm: from 431.60 (LD 0-4) to 943.35 mg/kg day (LD 14-21)

All reproductive parameters were equivalent across all groups, except that precoital interval was significantly longer (p<0.05) at 5000 ppm than at 0 ppm.

F1 Adult Female Terminal Necropsy
Terminal body weight of the F1 adult females were equivalent across all groups
F1 female adult absolute and relative organ weights were equivalent across groups except for:
- Absolute liver weights: significantly increased at 2500 (p<0.01) and 5000 ppm (p<0.001);
- Relative liver weights: significantly increased at 2500 (p<0.05) and 5000 ppm (p<0.001).
- Relative left ovary weight: significantly increased at 250 ppm (at p<0.05).

Vaginal cytology of the F1 females at necropsy indicated no differences among groups for the stage of Estrus (6-10/group), then Metestrus (5-10), then Diestrus (3-7 ), then Proestrus (1-3). Estrus stage could be determined in 2 females at 0 ppm, 0 at 250 ppm, 3 at 750 ppm, 1 at 2500 ppm and 4 at 5000 ppm.

F1 female gross pathology at terminal necropsy showed very few lesions and none that were treatment or dose related.

Findings from females terminated at scheduled necropsy included brown discoloration on the adrenal gland in one female at 0 ppm, right kidney dilation in one female at 750 and in two females each at 2500 ppm and 5000 ppm, and various lesions on the skin in all groups except the top dose group of 5000 ppm.
One F1 female from the control group was killed moribund. Only red discharge from vagina was recorded.
F1 female histopathology showed a very slight increase in bilateral cortical vacuolation of the adrenal glands only at 5000 ppm (2 females).
No other lesions including reproductive organ histopathology are possible treatment- or dose-related.
Dose descriptor:
NOAEL
Effect level:
750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased relative/absolute liver weight
Remarks on result:
other: Generation not specified (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced in males at PND14 onwards
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
F1 Litters
No differences in any parameter for F1 litter sizes across groups.
On PND 7 live females per litter were significantly increased (at p<0.05) and live males per litter were reduced, but not statistically significantly, both at 5000 ppm.
Percentage of males per litter was significantly reduced on PND 14 and on PND 21 (p<0.05) at 5000 ppm.
Live males per litter were also reduced on PND 21 (not statistically significantly.
Live females per litter were significantly increased (p<0.05) at 5000 ppm.
F1 male and female anogenital distances per litter on PND 0 were equivalent across all groups.
Overall mean pup body weight per litter was equivalent across 0-2500 ppm, but
significantly reduced at 5000 ppm on PND 0, unaffected across all dose groups on PND 4 and 7, significantly reduced at 2500 ppm (p<0.05) and at 5000 ppm (p<0.001) on PND 14, and significantly reduced at 5000 ppm (p<0.001) on PND 21 at weaning.
F1 male mean body weights per litter were significantly reduced at 5000 ppm on PND 0, unaffected on PND 4 and 7; significantly reduced at 250 ppm (p<0.05), at 2500 ppm (p<0.05) and at 5000 ppm (p<0.001) on PND 14; and significantly reduced at 5000 ppm (p<0.001) on PND 21. F1 female pup body weights per litter were unaffected across groups for PND 0, 4, and 7, and significantly reduced at 5000 ppm on PND 14 (p<0.05) and PND 21 (p<0.01).

F1 mean pup body weight changes (sexes combined) per litter were significantly reduced for PND 7-14 at 2500 and 5000 ppm (both at p<0.01), but not for PND 14-21. F1 mean male pup body weight changes per litter were reduced for PND 7-14 at 250 ppm (p<0.05), 750 ppm (p<0.05), and at 2500 and 5000 ppm (both at p<0.01). For PND 14-21, only the male pup body weight changes per litter at 5000 ppm were significantly reduced (at p<0.01).
F1 female pup body weight changes per litter were unaffected across groups for PND 0-4 and 4-7; they were significantly reduced at 2500 and 5000 ppm for PND 7-14 (both at p<0.01) and unaffected at any dose for PND 14-21.
No F1 pup clinical observations during lactation in the top two dose groups (2500 and 5000 ppm) were recorded.
F1 pup gross pathology findings were approximately equivalent in type and severity across all groups/sex.

For F1 male pups on PND21:
- Mean terminal body weight per litter: significantly reduced (at p<0.01) at 5000 ppm.
- Absolute, but not relative,brain, spleen and thymus weights: significantly reduced at 5000 ppm (at p<0.01).
- Relative (but not absolute) liver weight: significantly increased at 2500 ppm (p<0.01), but not at 5000 ppm.

For F1 female pups on PND 21:
- Terminal body weight: significantly reduced (at p<0.05) at 5000 ppm.
- Relative (but not absolute) liver weight: significantly increased at 2500 ppm (at p<0.01) but not at 5000 ppm.
- Spleen absolute and relative weights: reduced (at p<0.05) at 5000 ppm.

No apparent treatment- or dose-related gross findings on F1 pups terminated on PND 21 were recorded.



F2 Litter
Total litter size, live litter size, live birth and survive ratio from PND 0 to 21 were all statistically equivalent across all groups.
F2 male and female anogenital distances (AGD) per litter on PND 0 were all equivalent across groups.

Mean pup body weights, body pup body weight changes were also equivalent across all groups for all lactational intervals.
Pup clinical observations during lactation were all equivalent across groups in type of finding, incidence and severity of finding.

Gross pathology findings during lactation were all equivalent across groups for incidences, findings and severities.

All live F2 pups were euthanized on PND 21, weighed and subjected to necropsy, and external and visceral examination with pup organs weighed.

F2 male pup mean terminal body weights and organ weights by sex by litter were equivalent across all groups.
- For the F2 males:
-Relative liver weights: increased at 5000 ppm (p<0.001).
-Absolute spleen weight: significantly increased at 250 ppm (p<0.05) and 2500 ppm (p<0.05);
-Relative spleen weight: was significantly increased only at 250 ppm (p<0.05).

- F2 female pups: terminal body weights/litter were equivalent across all groups.
-Relative liver weight/litter: significantly increased at 2500 ppm (at p<0.05) and at 5000 ppm (at p<0.001).

F2 pup gross pathology findings were minimal; no gross findings nor gross lesions.
Only one female was found with a missing left eye at 750 ppm.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects upon reproduction (none observed)
Reproductive effects observed:
no

Table 1.

Mean values for abnormal sperm (%) in the 2 -generation study for S216A. HC1 and HC2 represent historical control values for studies conducted in the same lab using the same strain of rat (Cd(SD)) from the same breeder (Charles River) and published in the open literature. Lower rows give the SEM for n=25 (except HC2, n=30). All malformations noted in treated animals were one of two common findings in unteated animals. There were no unusual or unique malformations. * p<0.05, ** p<0.01, *** p<0.001.

Santicizer-216A in diet (ppm)
0 250 750 2500 5000 HC1 HC2
F0 (mean) 1.35 1.59 1.73* 1.95*** 2.16*** 3.29 2.12
F1 (mean) 1.45 1.72** 1.96*** 1.91*** 2.31*** 1.98 5.99
F0 (SEM) 0.07 0.08 0.06 0.11 0.17 0.92 0.21
F1 (SEM) 0.06 0.06 0.05 0.06 0.06 0.16 3.18
Conclusions:
The test material Santicizer® 261A (S-261A) presented a No Observable Adverse Effect Level (NOAEL) of 750 ppm (approximately 56 mg/kg/day S-261A for males and 50 mg/kg/day S-261A for females). There is no consistent evidence of reproductive toxicity for either males or females at dose levels.
Executive summary:

The two-generation study OECD 416 Guideline was performed on the test material Santicizer ® 261A (S-261A) in Sprague-Dawley Rats to study the integrity and performance of the male and female reproductive systems, including gonadal function, the oestrous cycle, mating behaviour, conception, gestation, parturition, lactation in the F0 and F1 generations and pre- and postnatal growth and development of the offspring in the F2 generation (to weaning). The animals were exposed to 750, 2500 and 5000 ppm of the test material in diet.

The test material is not a reproductive toxicant in either males or females. No effect on mating, fertility, fecundity, pup sex ratio/litter, or on developmental endocrine-mediated landmarks at any dose were related to the test material. The age at vaginal patency and preputial separation were unaffected as well as vestigial areolae or nipples in both F1 and F2 offspring.

The only finding in the offspring was reduced bodyweight in F1 males at birth and F1 males and females on postnatal days 14 and 21, when the animals started ingesting treated feed. However, these findings were not replicated in the F2 offspring and are not considered to represent a treatment-related effect on development. In addition, there was no effect upon male F1 bodyweight at birth following administration of S-261A at concentrations up to 7500ppm in the diet during an extended developmental toxicity study (Tyl et al 2005), further supporting the conclusion that birth weight is not affected by treatment with this substance.

A slight statistically-significant dose-related effect in the percentage of abnormal sperm was found in F0 adult males exposed to 750 ppm or more and in F1 adult males exposed to 250ppm and above. The malformations were limited to blunt hook (the most common finding) and head only/tail only (second most common finding) across all groups, including control males. There were no new sperm malformations observed across dose groups. The incidences of these findings were comparable to the historical control values for this rat strain and supplier; the incidence of abnormal sperm in F0 controls being 2.12+/-0.21% and 3.9+/-0.92%, and in F1 controls it is 5.99+/-3.18% and 1.98+/-0.16.

Based on the results of the study, the No Observable Adverse Effect Level (NOAEL) for effects upon reproduction is 5000ppm, which approximates to 375 mg/kg/day S-261A for males and 333 mg/kg/day S-261A for females. The overall NOAEL is 750 ppm, which approximates to 56 mg/kg/day S-261A for males and 50 mg/kg/day S-261A for females, based upon systemic toxicity (increased absolute and relative liver weight) at 2500ppm and 5000ppm.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-04-03 to 2018-08-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Modified according to ECHA Final Decision Letter dated 19 May 2017 (see background material)
Deviations:
yes
Remarks:
Study deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
SPECIES SELECTION:
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics and the historical control data available at Covance Harrogate.
BACKGROUND INFORMATION:
Non-GLP dietary 14-day study was performed with 1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters (Covance Study 8381227 [Covance Laboratories Ltd., 2018a])
Dietary OECD 414 study was performed with 1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters (RTI Study Code: Rt11-SAN1) (Tyl, 2013a)
Dietary OECD 416 study was performed with 1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters at a dose level of 250, 750, 2500, or 5000 ppm (Tyl, 2013b)

ROUTE OF EXPOSURE
The oral (dietary) route of administration was requested in the ECHA’s final decision letter, dated 19 May 2017; based on test article uses, this is a possible route of human exposure.
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its
reproductive characteristics and the historical control data available at Covance-Harrogate.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P): At start of dosing: 10 to 12 wks
- Weight at study initiation: (P) Males: 360.7 g and 487.9 g; Females: 229.9 g and 320.6 g
- Fasting period before study: Not specified
- Housing: in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes(Home Office, 2014). During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation and with their litter during the lactation phase. Males were returned to group housing after the pairing phase
- Diet (e.g. ad libitum): 5LF2/5KB3 EU Rodent Diet (International Product Supplies Ltd., London, United Kingdom), or LabDiet 5002 diet (Special Diets Services Ltd, Witham, United Kingdom) ad libitum
- Water (e.g. ad libitum): Water from the main tap supply was provided ad libitum via water bottles
- Acclimation period: at least 12 days of acclimation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): minimum of 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours of light and 12 hours of dark

IN-LIFE DATES: From: 2018-04-16 To: 2018-08-29
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
The control article (vehicle) was 5LF2/5KB3 EU Rodent diet
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): formulations were prepared weekly.
- Mixing appropriate amounts with (Type of food): The test article was formulated as a diet mix in 5LF2/5KB3 EU Rodent diet or LabDiet 5002 following dispensary SOPs and the formulation method as maintained in the study data.
- Storage temperature of food: Formulations were stored at room temperature (15 to 25C) in a sealed container.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The control article (vehicle) was 5LF2/5KB3 EU Rodent diet
Details on mating procedure:
- M/F ratio per cage: one male was housed for up to 10 days with one female of the same group.
- Length of cohabitation: up to 10 days
- Proof of pregnancy: Mating was confirmed by the presence of a vaginal plug in situor of sperm in a vaginal washing and referred to as day 0 of pregnancy.
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): Following mating, females were housed individually during gestation and with their litter during the lactation phase. Males were returned to group housing after the pairing phase.
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations prepared for use at the beginning (5LF2 EU rodent diet and Lab diet 5002) and end of the dosing period (5LF2 EU rodent diet) were analyzed to determine
the achieved concentration. The mean % nominal concentration should be between 85 to 115% and with a relative standard deviation (RSD) ≤10.0%. Results were within these criteria.Test article was not detected in the Group 1 control samples.The analytical procedure was validated Covance study 8381228.
Duration of treatment / exposure:
The test item was administered to animals for a maximum duration of approximately 98 or up to 65 days for males and females, respectively.
Frequency of treatment:
Continuous in diet
Details on study schedule:
Four groups of 20 rats/sex/group were administered 0 (control article), 375, 1500, or 6000 ppm of test article in 5LF2 EU or 5KB3 EU rodent diet, for a maximum duration of approximately 98 or up to 65 days for males or females, respectively. The control group was provided access to 5LF2 EU or 5KB3 EU Rodent Diet (expanded ground fine) or LabDiet 5002 ad libitum.
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
375 ppm
Remarks:
Low Concentration
Dose / conc.:
1 500 ppm
Remarks:
Intermediate Concentration
Dose / conc.:
6 000 ppm
Remarks:
High Concentration
No. of animals per sex per dose:
20/sex/concentration
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
Dose levels were selected based on a previous 14-day dietary study in rats (Covance Study 8381227 (Covance Laboratories Ltd., 2018a)) using dose levels of 250, 2500
and 7500 ppm 1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters.

A constant ratio of 4 was used to set doses after deciding the high dose as to be 6000 ppm (ie 6000, 1500, 375 ppm). The high dose level of 6000 ppm was expected to
produce evidence of toxicity based on the substantially lower mean body weight gain noted in females previously administered 7500 ppm. The low dose level of 375 ppm
was selected in the absence of significant findings at 250 ppm in the previous study, and was expected to be the minimum NOAEL. The intermediate dose level of 1500 ppm was designed to explore dose-relationship.

- Rationale for animal assignment (if not random):
Upon arrival, animals were assigned to dose groups using a total randomization procedure. Animals were individually identified by electronic implant. During the FOB assessments, applicable animals were identified by tail marks.

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at the beginning and end (nominal) of each working day for signs of ill health or overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes (Table 2)
- Time schedule: Each animal was given a detailed physical examination once during acclimation (Day 1 of the predose phase) and on the days of body weight recording.

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded once during acclimation (Day 1 of the predose phase), on the first day of dosing (Pre-Pairing Day 1) and at weekly intervals thereafter, and on the day prior to, and of, necropsy (Post-Pairing Day 13 and terminal sacrifice).

Female body weights were recorded during acclimation (Days 1 and 8 of the predose phase); on the first day of dosing (Pre-Pairing Day 1); weekly prior to pairing until the confirmation of mating; on Gestation Days (GD) 0, 7, 14, and 20; and on LD 1, 4, 7, 13, 21, and 22 (prior to necropsy).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumptions were
recorded for females from GD 0 to 20; and from LD 1 to 21. Consumption was calculated as g/animal/day.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

OTHER:
- Natural Delivery: Animals were observed three times/day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female had littered, or until a potential GD 25, whichever was earlier. Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation were recorded, where possible, and marked the end of gestation; when not observed; the end of gestation was the day when the completion of parturition was recorded or on the day prior to when LD 1 observations were made. Abnormal observations of nesting, parturition, or nursing were recorded. It was considered important that the dam was disturbed as little as possible on LD 0. Any dead pups were removed and sent to necropsy to establish if they were born alive or dead (by looking for lung inflation).

- Functional Observational Battery (FOB): All adult animals were assessed by detailed clinical observations, quantitative assessments, and for elicited responses. Males were assessed once prior to dose initiation and once weekly thereafter. Females were assessed once prior to dose initiation; once weekly during the pre-pairing and pairing phases; on GD 0, 7, 14, and 20; and on LD 1, 7, and 21.

The females which did not have a confirmation of mating and were assigned to the post-pairing phase, but then littered (Animals R0405, R0408, and R0412 [Group 1] and R0511 [Group 2]), had data removed from the post-pairing phase and entered into the gestation phase, on GD 12, 13, and 19.

Functional observational battery (FOB) assessments were ‘blinded’ so the observer did not know the dose group of animals during testing. Observations were performed at the same time in the day on each occasion.

Each animal was observed and evaluated weekly for the parameters listed in Table 2.

- Locomotor Activity: Locomotor activity was assessed in an automated photocell activity recorder for 30 minutes and was undertaken for five selected animals/sex/group (five males with the highest identification numbers and the first five littered females/group). Assessments were performed during the 14th week of dosing (Post-Pairing Day 7) for males and on LD 14 for females.

Activity counts were recorded at 5-minute intervals. The following parameters were determined: Total activity counts; Total mobile counts; Total rearing.

- Quantitative Assessments: Quantitative assessments were undertaken for selected animals/sex/group (ten males and the first six or seven littered females/group). Assessments were performed during the 14th week of dosing (Post-Pairing Day 8) for males and on LD 14 for females.

Quantitative assessment parameters are as follows: Hind limb foot splay; Fore and hind limb grip strength.

- Clinical Pathology: Blood samples for hematology (1 x 0.5 mL [EDTA], nominal), coagulation (1 x 0.5 mL [trisodium citrate], nominal), clinical chemistry (1 x 0.6 mL [lithium heparin], nominal), and bile acids (1 x 0.5 mL [serum separator tube], nominal) were withdrawn from the abdominal aorta at necropsy on Study Day 99 (Post-Pairing Day 14 for males) or on LD 22 for females. Samples were collected after animals
were fasted overnight.

Each sample for bile acids was gently inverted 10 times and was left to clot for 30 minutes stored at room temperature (15 to 25°C). Samples were analyzed at Covance.

Blood samples for thyroid hormone (2 x 0.6 mL [serum separator tubes], nominal) were withdrawn from the jugular vein of adult males prior to necropsy on Post-Pairing Day 14. Blood samples for thyroid hormone (1 x 1.2 mL [serum separator tubes], nominal) were withdrawn from the abdominal aorta of adult females at necropsy on LD 22. Samples were collected after animals were fasted overnight. Sampling was performed at a similar time on each occasion. Adult male samples, one
sample was used for analysis; one retained as a spare. Adult female samples were split into two equal aliquots of 0.6 mL; one sample was used for analysis, one was retained as a spare. The spare sample was retained in storage until report finalization.

Thyroid hormone sampling was performed at a similar time on each occasion (between 09:00 and 13:00); samples were protected from light until frozen storage. Each blood sample for thyroid hormone analysis was gently inverted 10 times and was stored at room temperature (15 to 25°C) and centrifuged at 2300g for 10 minutes at approximately 4°C. The resultant serum was separated, transferred to uniquely labeled amber polypropylene tubes, and frozen at <-20°C. Samples were centrifuged and aliquoted within 2 hours of collection, and were analyzed at Covance.

Hematology: Parameters evaluated are listed in Table 3.
Clinical Chemistry: Parameters evaluated are listed in Table 4.
Thyroid analysis: Parameters evaluated are listed in Table 5.
Oestrous cyclicity (parental animals):
Daily vaginal washings were conducted for all females during acclimation (predose), for 14 consecutive days prior to dosing and the stage of estrous recorded. Daily vaginal washings were taken from females from the start of dosing until the confirmation of mating, and on the morning of LD 22.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight, For each male, sperm number, motility, and velocity were recorded by Computer Assisted Sperm Analysis (CASA) from a sample of sperm from one epididymis and epididymis cauda. Each sample was prepared for microscopic evaluation of sperm morphology. Slides prepared from the first 10 surviving males of each group were read for morphological changes.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On PND 4, litters were culled to 10 pups/litter, with five pups/sex, when possible. Pups were selected randomly, with no bias as to size or clinical status. Culled pups
had their sex determined.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other.

Daily records of mortality and changes in litter sizes were maintained. Where possible, pups found dead or in a moribund condition underwent a macroscopic necropsy.

CLINICAL OBSERVATIONS
Each pup was given a detailed clinical examination daily from PND 1.

BODY WEIGHT
On PND 1, 4, 13, and 21, pup body weights were recorded.

ANOGENITAL DISTANCE
Anogenital distance of all pups was recorded on PND 4.

NIPPLE/ AREOLAE COUNT
The number of nipples/areolae for male pups was counted on PND 13.

GROSS EXAMINATION OF DEAD PUPS: Yes
Surplus pups culled on PND 4 (to standardize litter size) and pups sent to necropsy on PND 21 were sacrificed by an intraperitoneal injection of sodium pentobarbitone
(overdose). Once a suitable deep plane of anesthesia was established, major blood vessels were severed to exsanguinate the animal (decapitation for PND 4 pups).
Sacrifices were carried out in group order.

Surplus pups culled on PND 4 were discarded following blood sample collection and sex determination. Full macroscopic examinations were conducted for all decedents and one pup/sex/litter sacrificed on PND 21. External examinations for gross abnormalities, with particular attention to the external reproductive genitals, were conducted for all remaining pups sent to necropsy on PND 21.

Thyroid glands of pups sacrificed on PND 21 (one pup/sex/litter across groups) were weighed approximately 24 hours post-fixation and preserved in 10% neutral-buffered formalin. Testes, epididymides, seminal vesicle (with dorsolateral prostate), ventral prostate, levator ani/bulbocavernosus muscle (LABC), and bulbourethral glands of all male pups sacrificed on PND 21 were weighed and retained in relevant fixatives.

- Clinical Pathology:
Blood samples for thyroid hormone analysis from pups culled on PND 4 (1 x 0.6 mL [Serum Separator Tube], nominal) were withdrawn by decapitation from at least 2 pups per litter, additional pups from the same dose group were pooled together to obtain the sample volume. Samples were analyzed.

Pups sacrificed on PND 21 had blood samples (2 x 0.6 mL [Serum Separator Tube], nominal) for thyroid hormone analysis withdrawn by cardiac puncture to provide four samples, two samples from male pups and two samples from female pups for each litter, where possible. When required, additional pups of the same sex were used from the same litter to obtain the required blood volume. One sample per sex/litter were analyzed, the second sample per sex/litter were not analyzed in the first instance.

Pups sacrificed on PND 21 had blood samples (1 x 0.6 mL [lithium heparin], nominal) for clinical chemistry withdrawn by cardiac puncture, to provide two samples for each litter (one sample from one male and one sample from one female).

Thyroid hormone sampling was performed at a similar time on each occasion (between 09:00 and 13:00); samples were protected from light until frozen storage. Each blood sample for thyroid hormone analysis was gently inverted 10 times and was stored at room temperature (15 to 25°C) and centrifuged at 2300g for 10 minutes at approximately 4°C. The resultant serum was separated, transferred to uniquely labeled amber polypropylene tubes, and frozen at <-20°C. Samples were centrifuged and aliquoted within 2 hours of collection, and were analyzed at Covance.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Males were sacrificed on Day 99 of study (Post-Pairing Day 14), by isoflurane anaesthesia and after an overnight period without food. Sacrifices were carried out in controlled randomization order (one cage of animals from Group 1, 4, 2 then 3, then repeated). Once a suitable deep plane of anaesthesia was established, the major blood vessels were severed to exsanguinate the animal.

- Maternal animals: Females were sacrificed by isoflurane anaesthesia on LD 22 (those that achieved pregnancy) or 26 days post-coitum(the female that did not litter) after an overnight period without food. Once a suitable deep plane of anaesthesia was established, the major blood vessels were severed to exsanguinate the animal. Littered females were sacrificed in a controlled randomization order (i.e., females with the same day of mating/littering were necropsied together). The uterus of the apparently non-pregnant female (R0404 [Group 1]) was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.

GROSS NECROPSY
- Gross necropsy consisted of:
Females: Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.
Males: Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [6] were prepared for microscopic examination and weighed, respectively.

Organ weights, as indicated in Table 6 were recorded at each scheduled sacrifice, excluding the non-littered female. Organs were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. The tissues listed in Table 6 were obtained from all adult animals within each group. Tissues were retained in 10% neutral-buffered formalin, unless otherwise indicated.

All tissues denoted by ‘E’ in Table 6 from all adult animals were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.

Additional sections of testes and epididymides were also stained with periodic acid-Schiff (PAS) for spermatogenic staging for all males.
Postmortem examinations (offspring):
SACRIFICE
- Surplus pups culled on PND 4 (to standardize litter size) and pups sent to necropsy on PND 21 were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anaesthesia was established, major blood vessels were severed to exsanguinate the animal (decapitation for PND 4 pups). Sacrifices were carried out in group order.

Surplus pups culled on PND 4 were discarded following blood sample collection and sex determination. Full macroscopic examinations were conducted for all decedents and one pup/sex/litter sacrificed on PND 21. External examinations for gross abnormalities, with particular attention to the external reproductive genitals, were conducted for all remaining pups sent to necropsy on PND 21.

Thyroid glands of pups sacrificed on PND 21 (one pup/sex/litter across groups) were weighed approximately 24 hours post-fixation and preserved in 10% neutral-buffered formalin.

Testes, epididymides, seminal vesicle (with dorsolateral prostate), ventral prostate, levator ani/bulbocavernosus muscle (LABC), and bulbourethral glands of all male pups sacrificed on PND 21 were weighed and retained in relevant fixatives.

GROSS NECROPSY
- Full macroscopic examinations were conducted for all decedents and one pup/sex/litter sacrificed on PND 21. External examinations for gross abnormalities, with particular attention to the external reproductive genitals, were conducted for all remaining pups sent to necropsy on PND 21.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid glands of pups sacrificed on PND 21 (one pup/sex/litter across groups) were weighed approximately 24 hours post-fixation and preserved in 10% neutral-buffered formalin. Testes, epididymides, seminal vesicle (with dorsolateral prostate), ventral prostate, levator ani/bulbocavernosus muscle (LABC), and bulbourethral glands of all male pups sacrificed on PND 21 were weighed and retained in relevant fixatives.
Statistics:
Please see 'Any other information on materials and methods incl. tables' for information on statistics.
Reproductive indices:
Male Fertility and Reproductive Indices
Male mating index (%) = (Number of males mating with at least 1 female / Number of males cohabitated with at least 1 female) x 100
Male fecundity index (%) = (Number of males impregnating at least 1 female / Number of males mating with at least 1 female) x 100
Male fertility index (%) = (Number of males impregnating at least 1 female / Number of males cohabitated with at least 1 female) x 100

Female Fertility and Reproductive Indices
Female mating index (%) = Mated females/females cohabited (excluding females sacrificed during Cohabitation) x 100
Female fecundity index (%) = Pregnant females/mated females (excluding females with an undetermined pregnancy status) x 100
Female fertility index (%) = Pregnant females/females cohabited (excluding females sacrificed during Cohabitation or with an undetermined pregnancy status) x 100

Mean duration of gestation (days)
Mean number of implantation sites
Mean number of pups born
Mean number of pups alive PND 1
% male pups PND 1 (litter)
% male pups PND 1 (mean)
Mean number of pups alive PND 4 before culling
Mean number of pups culled PND 4
Mean number of pups alive PND 4 after culling
Mean number of pups alive PND 7
Mean number of pups alive PND 13
% Post-implantation survival index (litter)
% Post-implantation survival index (mean)
% Live birth index (litter)
Offspring viability indices:
% Live birth index (mean)
% Day 4 Viability Index (litter)
% Day 4 Viability Index (mean)
% Weaning index (litter)
% Weaning index (mean)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical observations noted were predominantly related to thinning or staining of the fur, sore or lesions on the skin, and raised hair or hair loss on various body parts of control or test article-treated animals.

Other observations were vocalization for one male each administered diets containing 375 (animal R0105 on pre-pairing days 15, 22, 29 and 36) or 6000 ppm (animal R0301 on pre-pairing Day 22); thin appearance for two males administered diets containing 6000 ppm (R0319, R0320), noted on a few occasions (pre-pairing Days 50 and 57); impaired mobility, transiently observed for one male (animal R0307) administered diet containing 6000 ppm commencing on Day 51 until Day 64 of pre-pairing; minimally to moderately damaged paw(s) for one male each administered diets containing 375 (animal R0110 on pairing days 1, 7 and 14 and post pairing Day 7) or 6000 ppm (animal R0302 on post-pairing Day 14); and one obese male (animal R0202) administered diet containing 1500 ppm at the end of pairing and into post pairing (pairing day 14 and post pairing Day 7).
Two females administered diet containing 375 ppm (Animal R0512 on Lactation Day 21 and Animal R0515 on Gestation Days 0 and 7) had one or both eyes protruding, and one female (Animal R0706) administered diet containing 6000 ppm had minimal excretion/discharge from the urogenital area on Gestation Day 15 however littering data for this female did not indicate a loss of implantations.

The clinical observations noted in the study were considered incidental as they were either present in controls and/or comparable with observations routinely noted at this laboratory for rats, infrequent, or showed no relationship to dose or trend over the duration of dosing.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male body weight gain when administered diet containing 6000 ppm throughout the dosing period was lower than the Controls (approximately 13% from pre-pairing day 1 to post pairing day 13, 154.8 g versus 177.1 g control value). However, statistical significance was not achieved, except pre-pairing days 1 to 36 where the body weight change was statistically significantly reduced by approximately 21%. These reduction in body weight gain by up to 21% compared to control was considered adverse effect of test article. Mean male body weight gain over the dosing period for males administered diets containing 375 or 1500 ppm was similar to the controls.

Body weight gain for females administered diet containing 6000 ppm was reduced by approx. 23% on pre-pairing day 1 to 15 and was considered adverse effect of test article due to lack of effects on food consumption. On GD 14 to 20 mean body weight gain was statistically increased for females (83.7 g compared with 71.6 g of control) administered diet containing 6000 ppm. These transient changes had no effect on the clinical condition of the adult females or the litters and was considered incidental and unrelated to the test article. The overall weight gain from pre-pairing day 1 to lactation day 21 was approximately 15% higher in females administered 6000 ppm compared to control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected in the study.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hemoglobin distribution width (HDW) was statistically significantly increased (3.06 g/dL) in males administered diet containing 6000 ppm, compared with controls (2.69 g/dL). This HDW increase occurred in the absence of effects on absolute reticulocyte counts and other RBC parameters, therefore, this is of uncertain mechanism and unlikely relationship to test article due to the absence of changes in other erythroid parameters.

Eosinophil counts were statistically significantly decreased in males administered diets containing 375, 1500, or 6000 ppm, compared with control. However, the group mean values were within the historical control data range (0.04 x 10^9/L to 0.30 x 10^9/L). The range of values were within or near the range of the controls and therefore considered most consistent with normal variability. The changes in eosinophil counts were therefore not test article-related.

Statistically significantly decreased hemoglobin (HB) and packed cell volume (PCV) were noted in females administered diet containing 6000 ppm. The mean and individual values were within the historical control range (hemoglobin 13.3 to 16.9 g/dL; PCV: 39.6 to 51.3%). These changes were considered not to be an adverse effect of the test article.

Statistically significantly increased WBC (10.3x10^9/L compared with 8.0x10^9/L of control) and large unstained cells (LUC) - (0.10 x 10^9/L compared with 0.05 x 10^9/L of control) were noted in females administered diet containing 6000 ppm. The mean and most individual values of WBC and LUC were within the historical control range (WBC = 4.4 to 14.6 10^9/L; LUC = 0.02 to 0.18 x 10^9/L). Test article-related statistically significantly increased neutrophil was noted in females administered diets containing 1500 or 6000 ppm (3.13 and 3.08 x 10^9/L compared with 2.16 x 10^9/L of control) and was outside the range of historical control data (0.36 to 2.59 x 10^9/L). In the absence of evidence of inflammation this finding was considered not to be adverse.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significantly increased cholesterol was noted in males and females administered diet containing 6000 ppm (males = 1.5 mmol/L compared with 1.2 mmol/L of control; females = 1.9 mmol/L compared with 1.5 mmol/L of control). Individual values of test article treated animals were near or within the range of concurrent control and also were within the historical control range (males = 1.2 to 2.6 mmol/L; females = 1.1 to 3.5 mmol/L). Therefore, the changes were considered not test article related.

Aspartate aminotransferase activity (AST) increased (110 IU/L compared with 94 IU/L of control) in females administered diet containing 6000 ppm. The mean and individual values were within the historical control range (58 to 160 IU/L). Additionally, the finding corroborated with microscopic liver changes, which were associated with an adaptive response. Thus, these findings were considered not adverse effects of the test article.

Individual females administered 1500 ppm (animals R0602, R0604, R0611, R0616) or 6000 ppm (animals R0702, R0706, R0711, R0713, R0716) had decreased globulin concentration with secondary increase in albumin:globulin ratio. Therefore, mean albumin:globulin ratio was statistically significantly increased (2.5 compared with 1.9 of control), and globulin was statistically significantly decreased (18 and 17 g/L, respectively compared with 21 g/L of control) in females administered 1500 or 6000 ppm. However, these changes were though uncertain relationship to test article were considered not adverse due to individual values were within the historical control range (albumin:globulin ratio: 1.1 to 3.4; globulin: 13 to 34 g/L).

Total protein decreased (57 g/L compared with 60 g/L of control) in females administered 6000 ppm. Individual values were within the concurrent control and the historical control range (total protein: 52 to 85 g/L). Thus, the changes were considered not adverse effects of the test article.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No specific clinical observations (FOB) were noted in test article-treated groups, compared with controls, and the findings were considered incidental as they were present in controls, infrequent, showed no relationship to dose, or were in single pups in a given group.

Quantitative assessments, including urine pools, fecal boli, latency, rears, foot splay, forelimb grip, and hindlimb grip, revealed no effects in males or females.

Locomotor activity, including total activity, mobility and rearing, was not affected in parent males (post-pairing) and females (lactation).
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):
Upon microscopic examination, test article-related findings were recorded for the liver, thyroid, and kidney.

In the liver, diffuse hepatocyte hypertrophy was noted in both sexes fed diets containing 6000 ppm. This finding was characterized by the presence of enlarged hepatocytes without consistent zonal pattern and correlated with the large appearance noted macroscopically and the increased organ weight. Hepatocyte hypertrophy is commonly observed as an adaptive response associated with the metabolism of xenobiotics or their metabolites (Greaves, 2012, Hallet al., 2012).

In the thyroid, an increased incidence and severity of follicular cell hypertrophy was recorded for both sexes fed diets containing 6000 ppm. This diffuse finding was characterized by increased height of the follicular epithelium and reduced amounts of colloid leading to decreased follicular diameter.

In the kidney, an increased incidence and severity of pigment was recorded for males fed diets containing 1500 or 6000 ppm. This finding was characterized by the presence of yellow-brown cytoplasmic droplets in renal tubular epithelial cells. In addition, there was a marginal increase in incidence and severity of hyaline droplets in males fed diets containing 6000 ppm.

No other test article-related microscopic findings were recorded. Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age at this laboratory.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Assessment:
The thyroid stimulating hormone (TSH) and thyroxine (T4) concentrations were not statistically significantly different in test article-treated groups, compared with control. However, dose-related increased TSH and dose-related decreased T4 concentrations were noted in both sexes administered diet containing 375, 1500 or 6000 ppm. In animals administered diet containing 6000 ppm, these changes corroborated with a microscopically increased incidence and severity of follicular cell hypertrophy in the thyroid gland, characterized by increased height of the follicular epithelium and reduced amounts of colloid leading to decreased follicular size. These findings were considered not adverse as the microscopic changes in the thyroid were considered adaptive changes and also secondary to diffuse hepatocyte hypertrophy. In the rat, thyroid follicular cell hypertrophy is generally considered an adaptive change due to increased thyroid hormone metabolism in the liver and is commonly associated with liver cell hypertrophy (McClain, 1989; Hardisty and Boorman, 1990; Maronpot and Brix; Curran and DeGroot, 1991; Ennulat et al., 2010).
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number and duration of estrous cycles was unaffected by dietary administration of the test article. Five animals in control and three, two, and four females administered diets containing 375, 1500, or 6000 ppm, respectively, had lower or higher than normal estrous cycle lengths. However, all affected females were pregnant and therefore this was considered incidental an unrelated to test article administration.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No test article-related findings were noted for sperm count, sperm motility, average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), straightness (VSL:VAP), or sperm morphology.
Reproductive performance:
no effects observed
Description (incidence and severity):
Male and female fertility and reproductive performance were not affected by dietary administration of the test article; mean values for the mating, fecundity, and fertility indices were similar to the controls.One control female (Animal R0404) was not pregnant, but all females were pregnant in test article-treated groups.

The number of females mated that were pregnant and delivered litters and the duration of gestation were not affected. The number of implantation sites and the
percentage of post-implantation loss were not affected. No test article-related effects were noted on the number of pups delivered, the number of live born, and number of still births occurred. Pup decedents were noted in controls and all test article-treated groups, but no dose-response relationship was noted and the decedents were not test article-related. Up to PND 21, pup survival indices were not affected.
No toxicologically significant changes were observed for reproductive toxicity/fertility endpoints assessed in the study.
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Remarks on result:
other: equivalent to 67 mg/Kg/day (males) and 151.83 mg/Kg/day (females)
Key result
Dose descriptor:
NOAEL
Effect level:
6 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
6 000 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations related to the skin/fur, including staining, thinning, and sores or lesions, were noted for a higher number of pups from litters of dams fed diets
containing 6000 ppm. These changes were considered non-specific or nonadverse effects of the test article.

One pup (Animal R0505-15 female) administered 375 ppm (on three occasions) and three pups (Animals R0713-15 female, R0717-14 female, and R0718-15 female) administered 6000 ppm (up to six occasions) were blue in colour; this is usually attributed to the nursing behavior of dams. In the absence of behavioral changes in dams, these findings were considered not test article related.

Other observations were considered incidental as they were present in controls, infrequent, and showed no relationship to dose.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of pups decedents was not dose-response related (17 pup from the control group; ten pups from the group administered 375 ppm; nine pups from the group administered 1500 ppm, and ten pups from the group administered 6000 ppm) or test article related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Male and female pup weights, measured on PND 1, PND 4 (pre- and post-cull), PND 13, and PND 21, and were not affected by test article administration; values were
similar to the controls.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Albumin:globulin ratio was increased in pups of the groups administered 1500 or 6000 ppm; this finding attained statistical significance for males of the group administered 6000 ppm (3.6 compared with 2.8 of control) and for females of groups administered 1500 or 6000 ppm for females (3.4 compared with 2.9 of control). Globulin decreased significantly (male and female pups of groups administered 1500 or 6000 ppm). The changes in globulin and albumin:globulin ratios affected individual animals and were consistent with individual animal variability and therefore were not test article-related.

Alkaline phosphatase and alanine aminotransferase activities were increased in both sexes of all test article-treated groups, compared with control. However, individual values were within or near the range of the concurrent controls and were consistent with individual animal variability and therefore were not test article-related.

Aspartate aminotransferase activity was increased in both sexes of the group administered 6000 ppm however the individual values were within or near the range of control animals and were consistent with individual animal variability and therefore were not test article-related.

Blood urea nitrogen and urea levels were statistically significantly increased for female pups of groups administered 375 or 6000 ppm. No dose-response relationship was observed and additionally, individual values were within or near the range of the controls and were consistent with individual animal variability and therefore were not test article-related.

Triglycerides were statistically significantly decreased in male pups of the group administered 1500 or 6000 ppm. However, individual values were within or near the range of the controls and were consistent with individual animal variability and therefore were not test article-related. In females, no dose response-related changes were noted.
Urinalysis findings:
not examined
Sexual maturation:
not specified
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance of male and female pups, measured on PND 4, was not affected by test article administration; values were similar to the controls.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
None of the male pups of control and test article-treated groups had nipples/areolae on PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related organ weight changes were recorded.

The apparent lower group mean bulbourethral absolute weights and body weight ratios for male pups of dams fed diets containing 1500 or 6000 ppm did not attain statistical significance and individual values were quite variable. For these reasons and in the absence of any other changes in primary or secondary male sex organ weights these minor differences were therefore considered within normal biological variability for this parameter and unrelated to test article.

None of the absolute or organ weight ratio changes were considered test article-related as they were small in magnitude, not dose-dependent, inconsistent between sexes, and/or due to normal inter-animal variability.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test article-related macroscopic findings were recorded.

Most tissues were macroscopically unremarkable, or the findings observed were generally consistent with the usual pattern of findings in rat pups of the same age and strain at this laboratory.
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
In male and female combined pups (PND 4), T4 and TSH levels were not affected. For pups measured by sex on PND 21, T4 levels were not affected, and TSH levels were increased in male pups of groups administered 1500 or 6000 ppm and in females pups of groups administered 375, 1500, or 6000 ppm. This increase in TSH was considered test article-related but was considered not adverse due to no corresponding or associated effects in T4 concentration and additionally no changes in thyroid weight.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
No toxicologically significant changes were observed for developmental endpoints assessed in the study.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
6 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Formulation Analysis:

Formulations prepared for use at the beginning and end of the dosing period were analyzed to determine the achieved concentration. The mean % nominal concentration of formulated diet were between accepted criteria of 85 to 115% and with a relative standard deviation (RSD) of ≤10.0%. Test article was not detected in Group 1 control samples.

Test Article Consumption:

During the pre-pairing phase, the average test article consumption for males was 18.2, 73.3, or 298.8 mg/kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the post-pairing phase, the average test article consumption was 15.7, 60.7, or 260.2 mg/kg/day for those administered 375, 1500, or 6000 ppm, respectively.

 

During the pre-pairing phase, the average test article consumption for females was 23.6, 99.2, or 367.3 mg/kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the gestation phase, the average test article consumption was 25.3, 106.2, or 410.7 mg/kg/day for those administered 375, 1500, or 6000 ppm, respectively, and during the lactation phase, the average test article consumption was 65.5, 250.1, or 974.7 mg/kg/day for those administered 375, 1500, or 6000 ppm, respectively.

Table 7. Summary of Body Weight Gain per Interval (Males)

Group

(Dose Level ppm)

 

Data Presented in "g" Interval X through X

Phase

Pre-pairing

Pre-pairing 1 -

Day

1-8

1-15

1-22

1-29

1-36

1-43

1-50

1-57

1-64

1-71

Pairing 7

Pairing 14

Post Pairing 7

Post Pairing 13

1

Control (0)

Mean

32.8

52.5

70.4

81.9

102.9

122.5

128.0

135.4

150.3

157.8

163.2

159.7

178.9

177.1

SD

15.26

20.96

20.95

26.08

26.90

30.01

34.26

31.42

37.13

42.57

39.33

39.35

41.73

38.23

N

20

20

20

20

20

20

20

20

20

20

20

20

 20

 20

 

2

(375)

Mean

29.0

46.5

64.9

77.1

96.8

104.8

119.0

129.7

144.2

155.4

156.7

163.5

176.5

173.4

SD

16.22

19.78

21.52

24.06

28.28

29.85

33.96

31.56

34.09

36.21

41.05

39.29

44.84

40.95

N

20

20

20

20

20

20

20

20

20

20

20

20

20 

 20

 

3

(1500)

Mean

33.7

54.8

71.7

86.4

103.9

116.5

126.7

142.7

154.0

166.5

165.7

163.7

183.6

180.7

SD

21.75

25.35

28.83

30.52

33.42

37.74

40.66

40.31

43.96

45.45

47.47

52.32

51.29

48.66

N

20

20

20

20

20

20

20

20

20

20

20

20

 20

 20

 

4

(6000)

Mean

26.4

44.0

59.0

70.7

81.7*

94.1

99.1

111.6

125.9

137.8

135.9

137.9

156.1

154.8

SD

11.32

15.00

14.85

14.18

16.20

20.23

40.12

27.45

28.70

33.36

33.44

40.24

32.89

31.96

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

 

Statistics

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

AT

* P<=0.05

** P<=0.01

*** P<=0.001

A = ANOVA and Dunnett's

T = Rank-transformed data

 

Table 8. Summary of Body Weight Gain per Interval (Females)

Group

(Dose Level ppm)

 

PA: 1-

PA: 8

PA: 8-

PA: 15

GD: 0- GD: 7

GD: 7- GD: 14

GD: 14- GD: 20

GD: 20- LA: 1

LA: 1- LA: 4

LA: 1- LA: 7

LA: 7- LA: 13

LA: 13- LA: 21

LA: 21- LA: 22

1

Control (0)

Mean

-1.7

7.9

23.8

27.9

71.6

-100.6

9.1

12.7

15.7

0.9

-32.0

SD

11.92

9.59

8.65

9.21

17.60

27.29

12.11

14.28

13.61

22.23

11.49

N

20

20

16

16

16

16

19

19

19

19

17

 

2

(375)

Mean

-0.2

5.9

21.2

28.3

76.8

-102.3

11.1

10.0

13.7

-0.7

-34.1

SD

5.65

6.43

7.21

6.64

12.26

17.42

14.79

13.77

15.81

12.17

9.02

N

20

20

20

20

20

20

20

20

20

20

14

 

3

(1500)

Mean

1.2

7.8

19.7

29.5

79.8

-101.2

7.3

15.8

12.5

-1.9

-35.9

SD

7.68

11.21

8.52

6.03

9.85

13.52

18.37

13.85

14.07

14.51

12.12

N

20

20

20

20

20

20

20

20

20

20

18

 

4

(6000)

Mean

0.9

3.8

18.9

33.3

83.7*H

-109.9

12.3

8.1

20.1

0.6

-38.5

SD

6.33

4.86

8.43

6.25

11.49

16.32

8.53

7.19

11.77

14.46

6.21

N

20

20

20

20

20

20

20

20

20

20

11

PA - Pre-pairing

GD - Gestation

LA - Lactation

*H = Dunnett Exact Homogeneous Test Significant: 0.05 level

Table 9. Summary of Hematology Results

Group

(Dose Level ppm)

Sex

Males

 

Females

Parameter

HDW

g/dL

E

109/L

MPV fL

HB

g/dL

PCV

%

WBC

109/L

N

109/L

LUC. 109/L

N%

%

PT

sec

Phase

Post Pairing

Post Pairing

Post Pairing

Lactation

Lactation

Lactation

Lactation

Lactation

Lactation

Lactation

Day

14

14

14

22

22

22

22

22

22

22

1

Control

(0)

Mean

2.69

0.15

7.4

15.4

47.6

8.0

2.16

0.05

27

24.0

SD

0.130

0.053

0.42

0.67

2.16

2.13

0.811

0.023

7.4

1.59

N

17

17

17

19

19

19

19

19

19

18

 

 

2

(375)

Mean

2.79

0.11*

7.7

15.7

48.5

8.6

2.51

0.06

29

23.5

SD

0.226

0.040

0.54

0.57

1.81

1.89

0.689

0.037

7.2

1.38

N

20

20

20

20

20

20

20

20

20

20

 

 

3

(1500)

Mean

2.82

0.11**

7.8*

15.6

48.4

9.2

3.13**

0.06

34*

23.0

SD

0.182

0.041

0.36

0.50

1.65

3.01

1.475

0.081

7.5

1.09

N

19

19

19

20

20

20

20

20

20

20

 

 

4

(6000)

Mean

3.06***

0.11*

7.4

14.9**

45.5**

10.3*

3.08*

0.10*

31

22.7**

SD

0.267

0.030

0.45

0.50

1.79

2.76

0.864

0.073

6.2

0.74

N

18

18

18

20

20

20

20

20

20

20

 

Statistics

AT

A

A

A

A

A

A

A

A

A

* P<=0.05

** P<=0.01

*** P<=0.001

A = ANOVA and Dunnett's

T = Rank-transformed data

HDW = Haemoglobin Distribution Width

E = Eosinophils

MPV = Mean Platelet Volume

HB = Haemoglobin

PCV = Packed Cell Volume

WBC = White Blood Cells

N = Neutrophils

LUC = Large Unstained Cells

N% = Neutrophils %

PT = Tox Prothrombin time

Table 10. Summary of Clinical Chemistry Results

Group

(Dose Level ppm)

Sex

Males

 

Females

Parameter

CHOL mmol/L

HCRE umol/L

ASTP

IU/L

CHOL mmol/L

TP

g/L

ALB

g/L

GLOB

g/L

A\G RATIO

GLUC mmol/L

Phase

Post Pairing

Post Pairing

Lactation

Lactation

Lactation

Lactation

Lactation

Lactation

Lactation

Day

14

14

22

22

22

22

22

22

22

1

Control

(0)

Mean

1.2

29

94

1.5

60

39

21

1.9

7.6

SD

0.23

3.0

14.3

0.36

3.6

2.9

2.9

0.38

1.75

N

20

20

19

19

19

19

19

19

19

 

2

(375)

Mean

1.3

26*

97

1.5

61

41

20

2.2

9.0*

SD

0.26

3.9

15.6

0.26

2.4

2.9

2.8

0.34

2.09

N

20

20

20

20

20

20

20

20

20

 

3

(1500)

Mean

1.4

28

104

1.7

60

42**

18**

2.5***

9.8***

SD

0.27

2.2

15.3

0.28

3.0

3.5

2.9

0.54

1.76

N

20

20

20

20

20

20

20

20

20

 

4

(6000)

Mean

1.5*

25**

110**

1.9***

57*

41

17***

2.5***

8.7

SD

0.28

3.0

15.2

0.28

2.5

2.0

2.6

0.46

1.38

N

20

20

20

20

20

20

20

20

20

 

Statistics

A

AT

A

A

A

A

A

AT

A

* P≤0.05

** P≤0.01

*** P≤0.001

A = ANOVA and Dunnett's

T = Rank-transformed data

CHOL: Total Cholesterol

HCRE: Enzymatic Creatinine

ASTP: Aspartate Aminotransferase

TP: Total Protein

ALB: Albumin

GLOB: Globulin

A / G: Albumin/Globulin Ratio

GLUC: Glucose

Table 11. Summary of Thyroid Hormone Analysis (Males)

Group

(Dose Level ppm)

Parameter

TSHI µIU/mL

IMT4 nmol/L

Phase

Post Pairing

Post Pairing

Day

14

14

1

Control

(0)

Mean

0.75

64

SD

0.561

11.5

N

20

11

 

2

(375)

Mean

0.76

63

SD

0.575

8.7

N

20

16

 

3

(1500)

Mean

1.04

61

SD

1.200

12.7

N

20

11

 

4

(6000)

Mean

1.46

54

SD

1.694

10.1

N

20

14

 

Statistics

AT

A

A = ANOVA and Dunnett's

T = Rank-transformed data

 

Table 12. Summary of Thyroid Hormone Analysis (Females)

Group

(Dose Level ppm)

Parameter

TSHI µIU/mL

IMT4 nmol/L

Phase

Lactation

Lactation

Day

22

22

1

Control

(0)

Mean

0.77

44

SD

0.466

10.3

N

12

12

 

2

(375)

Mean

0.81

48

SD

0.602

11.5

N

19

19

 

3

(1500)

Mean

1.16

43

SD

0.692

13.2

N

14

14

 

4

(6000)

Mean

1.47

37

SD

1.016

6.4

N

16

16

 

Statistics

AT

A

A = ANOVA and Dunnett's

T = Rank-transformed data

Table 13. Summary of Seminology Data

 

 

 

Group 1

(0 ppm)

Group 2

(375 ppm)

Group 3

(1500 ppm)

Group 4

(6000 ppm)

Statistics

Sperm Count (epididymis) (M/g)

Mean

916.2

841.0

1054.5

1030.1

P3

SD

192.57

173.59

296.53

226.17

N

20

20

20

16@

 

 

Sperm Motility (%)

Mean

86

86

82

87

P3

SD

11.3

7.6

19.9

5.3

N

20

20

20

20

 

 

Average Path Velocity (VAP) (μm/s)

Mean

154.3

159.1

150.9

157.6

P3

SD

23.08

16.84

17.07

17.5

N

20

20

20

20

 

 

Curvilinear Velocity (VCL) (μm/s)

Mean

265.6

276.0

264.5

279.8

P3

SD

36.38

28.90

34.85

31.43

N

20

20

20

20

 

 

Straight Line Velocity (VSL) (μm/s)

Mean

108.9

113.3

108.3

114.2

P3

SD

15.49

13.75

10.66

14.26

N

20

20

20

20

 

 

Straightness (VSL:VAP) (%)

Mean

70

70

71

70

P3

SD

3.9

2.9

5.1

2.1

N

20

20

20

20

 

 

Morphology – Abnormal Sperm (%)

Mean

0.9

0.4

0.3+r

0.2+r

P3

SD

1.00

0.65

0.68

0.41

N

20

20

20

20

*r = Wilcoxon Rank Sum Test Significant at 0.05 level

+r = Wilcoxon Rank Sum Test Significant at 0.01 level

#r = Wilcoxon Rank Sum Test Significant at 0.001 level

@ = Number examined reduced due to excluded data

P3 = Kruskal-Wallis and Wilcoxon

Table 14.Summary of Estrous Cycle Data

 

Control Group 1 (0 ppm)

Group 2 (375 ppm)

Group 3 (1500 ppm)

Group 4 (6000 ppm)

No. of Estrous Cycles

Mean Cycle Length (days)

No. of Estrous Cycles

Mean Cycle Length (days)

No. of Estrous Cycles

Mean Cycle Length (days)

No. of Estrous Cycles

Mean Cycle Length (days)

Predose

Mean

1.6

5.1

1.8

4.2

1.7

4.4

1.7

4.6

SD

0.8

2.83

0.5

0.49

0.7

0.77

0.7

1.34

n

20

17

20

20

20

18

20

18

Pre-Pairing

Mean

2.0

4.2

2.3

4.1

2.1

4.3

2.7

4.1

SD

1.1

1.12

0.9

0.37

0.8

0.93

0.6

0.51

n

20

17

20

18

20

20

20

20

Estrous cycle = from 1st Oestrus to the next non-consecutive stage of the same type

 

Table 15. Summary of Male Fertility and Reproductive Performance

Treatment Group

Control Group 1

(0 ppm)

Group 2

(375 ppm)

Group 3

(1500 ppm)

Group 4

(6000 ppm)

Total males

20

20

20

20

  Unscheduled Deaths Prior to Cohabitation

0

0

0

0

Males Cohabitated

20

20

20

20

  Unscheduled Deaths During Cohabitation

0

0

0

0

Males mating with at least 1 female

17

19

20

20

Males impregnating at least 1 female

16

19

20

20

Mating Index (%)

85

95

100

100

Fecundity Index (%)

94

100

100

100

Fertility Index (%)

80

95

100

100

 

Table 16. Summary of Female Fertility and Reproductive Performance

Treatment Group

Control Group 1

(0 ppm)

Group 2

(375 ppm)

Group 3

(1500 ppm)

Group 4

(6000 ppm)

Total Females

20

20

20

20

  Unscheduled Deaths Prior to Cohabitation

0

0

0

0

Females Cohabited

20

20

20

20

  Unscheduled Deaths During Cohabitation

0

0

0

0

Females Mated

20

20

20

20

Pregnant Females

19

20

20

20

Non Pregnant Females

1

0

0

0

Matings Per Day Periods Of Cohabitation

 

 

 

 

   Day 1

3

6

3

1

   Day 2

5

7

6

3

   Day 3

4

2

4

11

   Day 4

3

4

7

5

   Day 5

1

0

0

0

   Day 7

1

0

0

0

Mating Index (%)

100

100

100

100

Fecundity Index (%)

95

100

100

100

Fertility Index (%)

95

100

100

100

Table 17. Summary of Parturition and Litter Data

 

Dose Level

Control Group 1

(0 ppm)

Group 2

(375 ppm)

Group 3

(1500 ppm)

Group 4

(6000 ppm)

Females: Mated

N

19

20

20

20

               Pregnant

N

19

20

20

20

 

%

100

100

100

100

               Delivering

N

19

20

20

20

 

%

100

100

100

100

Duration of Gestation

Mean

22.8

22.8

22.9

22.7

SD

0.7

0.5

0.5

0.5

N

19

20

20

20

Females with Liveborn Pups

N

19

20

20

20

              Gestation Index

%

100

100

100

100

              with Stillborn Pups

N

0

4

2

2

 

%

0.00

20.00

10.00

10.00

Females with no Liveborn Pups

N

0

0

0

0

 

%

0.00

0.00

0.00

0.00

Pups Delivered

 

274

288

278

300

Mean

14.42

14.40

13.90

15.00

SD

3.93

2.11

2.17

2.45

N

19

20

20

20

         Liveborn

 

274

282

276

298

Mean

14.42

14.10

13.80

14.90

SD

3.93

2.31

2.21

2.45

         Stillborn

 

0

6

2

2

Mean

0.00

0.30

0.10

0.10

SD

0.00

0.73

0.31

0.31

         Uncertain

 

0

0

0

0

Implantation Sites

 

284

293

301

317

Mean

14.95

14.65

15.05

15.85

SD

3.79

2,01

2.65

2.23

N

19

20

20

20

Post-Implantation Loss

Mean

0.53

0.55

1.25

0.95

SD

0.61

0.94

2.38

1.19

%

5.68

3.90

7.11

6.02

Females: Delivering Live Pups

 

19

20

20

20

  Died or Killed During Lactation

 

0

0

0

0

  Removed due to Total Litter Loss

 

0

0

0

0

  Surviving to Scheduled Sacrifice

 

19

20

20

20

Pup Survival Indices

 

 

 

 

 

       Livebirth Index

Mean%

100

98

99

99

       Day 4 Viability Index

Mean%

95

99

98

98

       Weaning Index

Mean%

100

92

95

100

Pup Disposition

 

 

 

 

 

       Culled day 4 or 21

TOTAL

79

95

80

91

       Dead Pup

 

11

1

3

4

       Moribund Pup Sacrifice

 

4

0

0

0

       Cannibalized

 

0

0

1

0

       Missing

 

2

3

3

4

Pups Surviving at 21 days

TOTAL

178

183

189

199

Pups Dead Pup, Moribund Pup Sacrifice, Missing and/or Cannibalized+

 

 

 

 

 

       Days 0-4

 

17

3

6

7

       Days 5-21

 

0

1

1

1

Entire Litter Dead Pup, Moribund Pup Sacrifice, Missing and/or Cannibalized+    

 

 

 

 

 

       Days 0-4

N

0

0

0

0

       Days 5-21

N

0

0

0

0

N = Number of Females or Litters

TOTAL = Number of Pups or Implants

+ Excludes Pups where the dam has died or was killed moribund

Table 18. Summary of Pup Survival

 

Dose Level

Control Group 1

(0 ppm)

Group 2

(375 ppm)

Group 3

(1500 ppm)

Group 4

(6000 ppm)

Total Number and Mean Males Percent by Litter

 

  Day 1

 

139

156

130

161

Mean%

49

57

48

56

  Day 4 Precull

 

130

156

129

160

Mean%

48

57

48

56

  Day 4 Postculla

 

87

109

101

107

Mean%

46

55

51

54

  Day 13a

 

87

109

101

106

Mean%

46

55

51

53

    Day 21

 

87

99

95

106

Mean%

46

52

50

53

Live Pups/Litter with Live Pups

 

    Day 1

 

N

19

20

20

20

Mean

14.26

14.05

13.60

14.60

SD

3.87

2.24

2.33

2.39

    Day 4 Precull

 

N

19

20

20

20

Mean

13.53

13.95

13.50

14.55

SD

3.91

2.14

2.24

2.35

    Day 4 Postcull

N

19

20

20

20

Mean

9.37

10.00

10.00

10.00

SD

2.14

0.00

0.00

0.00

    Day 13

N

19

20

20

20

Mean

9.37

10.00

10.00

9.95

SD

2.14

0.00

0.00

0.22

    Day 21

N

19

19

19

20

Mean

9.37

9.63

9.95

9.95

SD

2.14

1.38

0.23

0.22

N = Number of Litters

a No statistical analysis performed

Table 19. Summary of Pup Body Weights

 

Dose Level

Control Group 1

(0 ppm)

Group 2

(375 ppm)

Group 3

(1500 ppm)

Group 4

(6000 ppm)

Pup Weight/Litter (grams)

 

  Day 1 Males

Mean

7.03

7.09

7.25

6.90

SD

0.59

0.73

0.58

0.6

N

18

20

20

20

            Covariate adjusted

Mean

7.11

7.06

7.15

6.96

  Day 1 Females

Mean

6.69

6.71

7.00

6.59

SD

0.66

0.78

0.52

0.63

N

19

20

20

20

             Covariate adjusted

Mean

6.69

6.70

6.92

6.66

  Day 4 MALES - Precull

Mean

9.99

10.28

10.41

9.87

SD

0.85

1.07

0.93

1.16

N

18

20

20

20

             Covariate adjusted

Mean

10.11

10.24

10.26

9.95

 Day 4 FEMALES - Precull

Mean

9.41

9.66

10.00

9.41

SD

0.91

1.14

0.92

1.14

N

19

20

20

20

            Covariate adjusted

Mean

9.41

9.66

9.91

9.51

 Day 4 MALES - Postcull

Mean

9.99

10.27

10.41

9.86

SD

0.85

1.07

0.93

1.17

N

18

20

20

20

 Day 4 FEMALES - Postcull

 

Mean

9.41

9.67

10.01

9.40

SD

0.91

1.13

0.91

1.14

N

19

20

20

20

 Day 13 MALES

Mean

29.60

29.89

29.65

29.33

SD

2.00

2.32

2.03

2.92

N

18

20

20

20

 Day 13 FEMALES

Mean

27.99

28.78

28.77

28.42

SD

1.81

1.99

2.03

3.07

N

19

20

20

20

 Day 21 MALES

Mean

52.18

52.93

52.21

50.56

SD

3.42

4.32

4.98

6.07

N

18

20

19

20

 Day 21 FEMALES

Mean

49.93

50.97

50.56

48.52

SD

2.76

3.83

4.44

5.88

N

18

20

19

20

N = Number of Litters

Table 20. Summary of Pup Clinical Chemistry

Group

(Dose Level ppm)

 

Males

Females

A/G Ratio

AST (IU/L)

GLOBULIN (g/L)

TRIGLYCERIDES

(mmol/L)

Blood Urea

Nitrogen (mg/dL)

GLOBULIN (g/L)

UREA (mmol/L)

1

Control

(0)

Mean

2.9

109

12

1.16

13.3

12

4.8

SD

0.63

16.2

2.0

0.645

2.37

2.1

0.85

N

18

18

18

18

18

18

18

 

2

(375)

Mean

3.0

113

12

0.97

15.1*

11

5.4*

SD

0.52

36.3

1.8

0.493

2.53

1.2

0.91

N

20

20

20

20

20

20

20

 

3

(1500)

Mean

3.4*

109

10*

0.66*

13.8

10**

4.9

SD

0.83

10.6

1.9

0.304

2.33

1.7

0.83

N

20

20

20

20

20

20

20

 

4

(6000)

Mean

3.4*

113*

10*

0.59**

15.4*

10**

5.5*

SD

0.49

5.4

2.2

0.237

1.84

1.6

0.66

N

20

20

20

20

20

20

20

 

Statistics

A

AT

A

AT

A

A

A

* P<=0.05

** P<=0.01

*** P<=0.001

A = ANOVA and Dunnett's

T = Rank-transformed data

Table 21. Summary of Pup Thyroid Hormone Analysis (Male and Female pups combined) - PND 4

Group

(Dose Level ppm)

 

IMT4 nmol/L

TSH µIU/mL

1

Control

(0)

Mean

<19

0.27

SD

3.2

0.086

N

16

16

 

2

(375)

Mean

<19

0.27

SD

2.9

0.097

N

16

16

 

3

(1500)

Mean

<17

0.29

SD

2.9

0.139

N

16

16

 

4

(6000)

Mean

<19

0.27

SD

3.1

0.114

N

19

19

Table 22. Summary of Pup Thyroid Hormone Analysis - PND 21

Group

(Dose Level ppm)

 

Males

Females

IMT4 nmol/L

TSH µIU/mL

IMT4 nmol/L

TSH µIU/mL

1

Control

(0)

Mean

46

0.25

48

0.22

SD

7.7

0.128

12.8

0.115

N

18

18

18

18

 

2

(375)

Mean

45

0.27

43

0.28

SD

6.6

0.174

8.0

0.143

N

20

20

18

18

 

3

(1500)

Mean

46

0.35

43

0.31

SD

11.3

0.238

10.5

0.205

N

20

20

20

20

 

4

(6000)

Mean

47

0.40

46

0.30

SD

10.6

0.261

11.3

0.185

N

                 20         

20

20

20

 

Statistics

AT

AT

A

A

PND = Postnatal Day

A = ANOVA and Dunnett's

T = Rank-transformed data

Conclusions:
Daily oral dietary administration of 375, 1500, or 6000 ppm of 1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters to groups of male and female Crl:CD (Sprague Dawley [SD]) rats for 98 days (males during pre-pairing, pairing, and post pairing phases) or up to 65 days (females during pre-pairing, pairing, gestation, and lactation phases) resulted in reduction in body weight gain for males and females administered 6000 ppm; an increased incidence of large appearance of liver for females administered 6000 ppm and increased group mean absolute and relative (to body weight) liver weights for animals administered 6000 ppm, compared with concurrent controls.
 
Increased haemoglobin distribution width (HDW) concentrations in males administered 6000 ppm was considered uncertain mechanism and unlikely to be related to the test material. Treatment-related microscopic findings were observed in the liver and thyroid of animals administered 6000 ppm and were considered to be adaptive rather than treatment-related. The increased pigment in the kidneys for males administered 1500 or 6000 ppm was likely to be non-adverse in the absence of any tubular degeneration, necrosis or inflammation.
 
Based on the results of this study, dietary exposure of 1500 ppm (males = 67 mg/Kg/day; females = 151.83 mg/Kg/day) is considered to be the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the F0 generation.
 
The NOAEL for reproductive toxicity in the F0 generation and developmental toxicity in the F1 generation pups was determined to be 6000 ppm since there were no toxicologically significant changes observed in the endpoints assessed in the study.
Executive summary:

A key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat was conducted. Repeated dose administration of the test material (1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters; CAS# 68515-40-2) was evaluated for systemic effects and to screen for effects on male and female reproductive performance (i.e. gonadal function, mating behavior, conception, development of the conceptus and parturition), and offspring growth until Lactation Day 21.

 

Four groups of Crl:CD(SD) rats (20/sex/concentration) were administered 0 (control article), 375, 1500, or 6000 ppm of test material in diet, for a duration of 98 days for males or up to 65 days for females. The control group was provided access to basal diet ad libitum.

 

Adults were observed for clinical effects, body weight, food consumption, motor activity, grip strength and foot splay, estrous cycling, mating, fertility and pregnancy indices, and offspring parameters. Pup clinical observations, litter size, sex, and body weights were recorded. Anogenital distance was recorded on Postnatal Day (PND) 4, and nipple retention was also recorded for male pups on PND 13. Complete necropsies were performed on all animals, and any macroscopic abnormalities were noted. Blood samples for hematology, clinical chemistry, bile acids and thyroid hormone assessments were collected at necropsy from all adults. Additionally, samples were collected at necropsy from selected PND 21 pups for clinical chemistry, and PND 4 and 21 pups for thyroid hormone assessments, thyroid weight recording and macroscopic examination. Organ weights were recorded and microscopic examinations were performed.

 

No test material-related mortality or clinical signs of toxicity were observed. Reduction in body weight gain by up to 21% and 23% was observed in males and females, respectively administered diet containing 6000 ppm. This was considered to be adverse and treatment-related. Food consumption was not affected by oral exposure to the test material.

 

During the pre-pairing phase, the average test material consumption for males was 18.2, 73.3, or 298.8.26 mg/Kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the pre-pairing phase, the average test material consumption for females was 23.6, 99.2, or 367.3 mg/Kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the post-pairing phase, the average test material consumption for males was 15.7, 60.7, or 260.2 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively. During the gestation phase, the average test material consumption was 25.3, 106.2, or 410.7 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively, and during the lactation phase, the average test material consumption was 65.5, 250.1, or 974.7 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively.

 

There were no adverse findings in either sex during the functional observational battery and locomotor activity assessments.

 

No effects on clinical chemistry parameters were observed in parental animals. Haematology results revealed statistically significantly increased haemoglobin distribution width (HDW) in males administered 6000 ppm. In the absence of changes in red blood cell count (RBC), hemoglobulin (HB), mean corpuscular volume (MCV), or red cell distribution width (RDW), compared with control, the change in HDW was not considered not to be an adverse treatment-related effect. Statistically significantly increased neutrophil counts were observed in females administered 1500 or 6000 ppm and was considered treatment-related. However, in the absence of evidence of inflammation, this finding was not considered to be adverse.

 

At necropsy, treatment-related findings were observed in the liver. An increased incidence of large appearance was recorded for females fed diets containing 6000 ppm. No other macroscopic findings considered related to the test material were recorded. Other tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age at the laboratory.

 

Absolute and relative liver weights were higher for both sexes receiving 6000 ppm, attaining statistical significance (p<0.01 to p<0.001) with an increased magnitude of change observed in the females (approximately 35%), which also correlated with an increased incidence of large appearance macroscopically in females and microscopically in both sexes with diffuse hepatocyte hypertrophy, characterized by the presence of enlarged hepatocytes without consistent zonal pattern in the liver. This was associated with an increased incidence and severity of follicular cell hypertrophy in the thyroid gland, characterized by increased height of the follicular epithelium and reduced amounts of colloid leading to decreased follicular size, accompanied with decreased T4 levels (approximately 16% of control), and increased TSH levels (approximately 90% of control), respectively. The liver changes were also accompanied with increased aspartate aminotransferase activity in female rats administered 6000 ppm. Dose-related increased TSH and dose-related decreased T4 concentrations were observed in both sexes administered diet containing 375, 1500 or 6000 ppm and considered to be the result of adaptive changes in the thyroid gland and secondary to hepatocyte hypertrophy. Kidney weights (both absolute and relative) were increased for males given 6000 ppm, attaining statistical significance (p<0.001 to p<0.05, respectively), which also correlated microscopically with an increased incidence and severity of pigment for males given 1500 ppm or 6000 ppm. However, the increased weight and pigment in the kidneys for males administered 1500 or 6000 ppm was considered to be non-adverse in the absence of any associated tubular degeneration, necrosis or inflammation.

 

Male and female fertility and reproductive performance were not affected following oral dietary administration of the test material. The number of mated pregnant, the number of females delivering, and the duration of gestation were unaffected. The number of implantation sites and the percentage of post-implantation loss was also not affected. No test material-related effect on the number of pups delivered, the number of live born, and number of still births was observed post treatment. Pup survival indices were not affected up to PND 21. No test material-related pup mortality was observed. Male and female pup weights, measured on PND 1, PND 4 (pre- and post-cull), PND 13, and PND 21, were not statistically significantly affected in any group. Anogenital distance was not affected in male or female pups and none of the male pups had nipples/areolae.

 

No test material-related clinical chemistry changes were observed in pups. The thyroid stimulating hormone (TSH) level was increased in male pups of the groups administered 1500 or 6000 ppm and in females pups of the groups administered 375, 1500, or 6000 ppm on PND 21. This increase in TSH was considered treatment-related but not adverse due to no corresponding or associated effects in T4 concentration. Additionally, no changes in thyroid weight were observed. No effect on thyroid hormone was observed for PND 4 pups. No treatment-related macroscopic findings were observed for pups.

 

In conclusion, daily oral dietary administration of 375, 1500, or 6000 ppm of 1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters to groups of male and female Crl:CD (Sprague Dawley [SD]) rats for 98 days (males during pre-pairing, pairing, and post pairing phases) or up to 65 days (females during pre-pairing, pairing, gestation, and lactation phases) resulted in reduction in body weight gain for males and females administered 6000 ppm; an increased incidence of large appearance of liver for females administered 6000 ppm and increased group mean absolute and relative (to body weight) liver weights for animals administered 6000 ppm, compared with concurrent controls.

 

Increased haemoglobin distribution width (HDW) concentrations in males administered 6000 ppm was considered uncertain mechanism and unlikely to be related to the test material. Treatment-related microscopic findings were observed in the liver and thyroid of animals administered 6000 ppm and were considered to be adaptive rather than treatment-related. The increased pigment in the kidneys for males administered 1500 or 6000 ppm was likely to be non-adverse in the absence of any tubular degeneration, necrosis or inflammation.

 

Based on the results of this study, dietary exposure of 1500 ppm (males = 67 mg/Kg/day; females = 151.83 mg/Kg/day) is considered to be the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the F0 generation.

 

The NOAEL for reproductive toxicity in the F0 generation and developmental toxicity in the F1 generation pups was determined to be 6000 ppm since there were no toxicologically significant changes observed in the endpoints assessed in the study.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
333 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The database is of high quality, being based on a study conducted to the recognised OECD TG 416 and under GLP.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The two-generation study OECD 416 Guideline was performed on the test material Santicizer ® 261A (S-261A) in Sprague-Dawley Rats to study the integrity and performance of the male and female reproductive systems, including gonadal function, the oestrous cycle, mating behaviour, conception, gestation, parturition, lactation in the F0 and F1 generations and pre- and postnatal growth and development of the offspring in the F2 generation (to weaning). The animals were exposed to 750, 2500 and 5000 ppm of the test material in diet.

The test material is not a reproductive toxicant in either males or females. No effect on mating, fertility, fecundity, pup sex ratio/litter, or on developmental endocrine-mediated landmarks at any dose were related to the test material. The age at vaginal patency and preputial separation were unaffected as well as vestigial areolae or nipples in both F1 and F2 offspring.

A slight statistically-significant dose-related effect in the percentage of abnormal sperm was found in F0 adult males exposed to 750 ppm or more and in F1 adult males exposed to 250ppm and above. The malformations were limited to blunt hook (the most common finding) and head only/tail only (second most common finding) across all groups, including control males. There were no new sperm malformations observed across dose groups. The incidences of these findings were comparable to the historical control values for this rat strain and supplier; the incidence of abnormal sperm in F0 controls being 2.12+/-0.21% and 3.9+/-0.92%, and in F1 controls it is 5.99+/-3.18% and 1.98+/-0.16.

Based on the results of the study, the No Observable Adverse Effect Level (NOAEL) for effects upon reproduction is 5000ppm, which approximates to 375 mg/kg/day S-261A for males and 333 mg/kg/day S-261A for females. The overall NOAEL is 750 ppm, which approximates to 56 mg/kg/day S-261A for males and 50 mg/kg/day S-261A for females, based upon systemic toxicity (increased absolute and relative liver weight) at 2500ppm and 5000ppm.

A key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat was conducted (Covance Laboratories Ltd., 2019; Klimisch score = 1). Repeated dose administration of the test material (1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters; CAS# 68515-40-2) was evaluated for systemic effects and to screen for effects on male and female reproductive performance (i.e. gonadal function, mating behavior, conception, development of the conceptus and parturition), and offspring growth until Lactation Day 21.

 

Four groups of Crl:CD(SD) rats (20/sex/concentration) were administered 0 (control article), 375, 1500, or 6000 ppm of test material in diet, for a duration of 98 days for males or up to 65 days for females. The control group was provided access to basal diet ad libitum.

 

Adults were observed for clinical effects, body weight, food consumption, motor activity, grip strength and foot splay, estrous cycling, mating, fertility and pregnancy indices, and offspring parameters. Pup clinical observations, litter size, sex, and body weights were recorded. Anogenital distance was recorded on Postnatal Day (PND) 4, and nipple retention was also recorded for male pups on PND 13. Complete necropsies were performed on all animals, and any macroscopic abnormalities were noted. Blood samples for hematology, clinical chemistry, bile acids and thyroid hormone assessments were collected at necropsy from all adults. Additionally, samples were collected at necropsy from selected PND 21 pups for clinical chemistry, and PND 4 and 21 pups for thyroid hormone assessments, thyroid weight recording and macroscopic examination. Organ weights were recorded and microscopic examinations were performed.

 

No test material-related mortality or clinical signs of toxicity were observed. Reduction in body weight gain by up to 21% and 23% was observed in males and females, respectively administered diet containing 6000 ppm. This was considered to be adverse and treatment-related. Food consumption was not affected by oral exposure to the test material.

 

During the pre-pairing phase, the average test material consumption for males was 18.2, 73.3, or 298.8.26 mg/Kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the pre-pairing phase, the average test material consumption for females was 23.6, 99.2, or 367.3 mg/Kg/day for those administered 375, 1500, or 6000 ppm, respectively. During the post-pairing phase, the average test material consumption for males was 15.7, 60.7, or 260.2 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively. During the gestation phase, the average test material consumption was 25.3, 106.2, or 410.7 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively, and during the lactation phase, the average test material consumption was 65.5, 250.1, or 974.7 mg/Kg/day for rats administered 375, 1500, or 6000 ppm, respectively.

 

There were no adverse findings in either sex during the functional observational battery and locomotor activity assessments.

 

No effects on clinical chemistry parameters were observed in parental animals. Haematology results revealed statistically significantly increased haemoglobin distribution width (HDW) in males administered 6000 ppm. In the absence of changes in red blood cell count (RBC), hemoglobulin (HB), mean corpuscular volume (MCV), or red cell distribution width (RDW), compared with control, the change in HDW was not considered not to be an adverse treatment-related effect. Statistically significantly increased neutrophil counts were observed in females administered 1500 or 6000 ppm and was considered treatment-related. However, in the absence of evidence of inflammation, this finding was not considered to be adverse.

 

At necropsy, treatment-related findings were observed in the liver. An increased incidence of large appearance was recorded for females fed diets containing 6000 ppm. No other macroscopic findings considered related to the test material were recorded. Other tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age at the laboratory.

 

Absolute and relative liver weights were higher for both sexes receiving 6000 ppm, attaining statistical significance (p<0.01 to p<0.001) with an increased magnitude of change observed in the females (approximately 35%), which also correlated with an increased incidence of large appearance macroscopically in females and microscopically in both sexes with diffuse hepatocyte hypertrophy, characterized by the presence of enlarged hepatocytes without consistent zonal pattern in the liver. This was associated with an increased incidence and severity of follicular cell hypertrophy in the thyroid gland, characterized by increased height of the follicular epithelium and reduced amounts of colloid leading to decreased follicular size, accompanied with decreased T4 levels (approximately 16% of control), and increased TSH levels (approximately 90% of control), respectively. The liver changes were also accompanied with increased aspartate aminotransferase activity in female rats administered 6000 ppm. Dose-related increased TSH and dose-related decreased T4 concentrations were observed in both sexes administered diet containing 375, 1500 or 6000 ppm and considered to be the result of adaptive changes in the thyroid gland and secondary to hepatocyte hypertrophy. Kidney weights (both absolute and relative) were increased for males given 6000 ppm, attaining statistical significance (p<0.001 to p<0.05, respectively), which also correlated microscopically with an increased incidence and severity of pigment for males given 1500 ppm or 6000 ppm. However, the increased weight and pigment in the kidneys for males administered 1500 or 6000 ppm was considered to be non-adverse in the absence of any associated tubular degeneration, necrosis or inflammation.

 

Male and female fertility and reproductive performance were not affected following oral dietary administration of the test material. The number of mated pregnant, the number of females delivering, and the duration of gestation were unaffected. The number of implantation sites and the percentage of post-implantation loss was also not affected. No test material-related effect on the number of pups delivered, the number of live born, and number of still births was observed post treatment. Pup survival indices were not affected up to PND 21. No test material-related pup mortality was observed. Male and female pup weights, measured on PND 1, PND 4 (pre- and post-cull), PND 13, and PND 21, were not statistically significantly affected in any group. Anogenital distance was not affected in male or female pups and none of the male pups had nipples/areolae.

 

No test material-related clinical chemistry changes were observed in pups. The thyroid stimulating hormone (TSH) level was increased in male pups of the groups administered 1500 or 6000 ppm and in females pups of the groups administered 375, 1500, or 6000 ppm on PND 21. This increase in TSH was considered treatment-related but not adverse due to no corresponding or associated effects in T4 concentration. Additionally, no changes in thyroid weight were observed. No effect on thyroid hormone was observed for PND 4 pups. No treatment-related macroscopic findings were observed for pups.

 

In conclusion, daily oral dietary administration of 375, 1500, or 6000 ppm 1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters to groups of male and female Crl:CD (Sprague Dawley [SD]) rats for 98 days (males during pre-pairing, pairing, and post pairing phases) or up to 65 days (females during pre-pairing, pairing, gestation, and lactation phases) resulted in reduction in body weight gain for males and females administered 6000 ppm; an increased incidence of large appearance of liver for females administered 6000 ppm and increased group mean absolute and relative (to body weight) liver weights for animals administered 6000 ppm, compared with concurrent controls.

 

Increased haemoglobin distribution width (HDW) concentrations in males administered 6000 ppm was considered uncertain mechanism and unlikely to be related to the test material. Treatment-related microscopic findings were observed in the liver and thyroid of animals administered 6000 ppm and were considered to be adaptive rather than treatment-related. The increased pigment in the kidneys for males administered 1500 or 6000 ppm was likely to be non-adverse in the absence of any tubular degeneration, necrosis or inflammation.

 

Based on the results of this study, dietary exposure of 1500 ppm (males = 67 mg/Kg/day; females = 151.83 mg/Kg/day) is considered to be the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the F0 generation.

 

The NOAEL for reproductive toxicity in the F0 generation and developmental toxicity in the F1 generation pups was determined to be 6000 ppm since there were no toxicologically significant changes observed in the endpoints assessed in the study.


Justification for selection of Effect on fertility via oral route:
This is are well-conducted studies to OECD 416, OECD 422 and GLP

Effects on developmental toxicity

Description of key information

In a prenatal developmental toxicity study (OECD 414) in Sprague-Dawley rats, S-216A fed in the diet at up to 3750ppm (ca. 250mg/kg bw/d) was without effect on

development.

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422), repeated administration of the test material (1,2-Benzenedicarboxylic acid, benzyl C7-9-branched and linear alkyl esters; CAS# 68515-40-2) in diet to groups of Crl:CD(SD) rats (20/sex/concentration) at concentrations of 0 (control article), 375, 1500, or 6000 ppm, for a duration of 98 days (for males) or up to 65 days (for females) resulted in no toxicologically significant changes observed in the developmental toxicity endpoints assessed in the study. The NOAEL for developmental toxicity in the F1 generation pups was determined to be 6000 ppm, the highest concentration tested (Covance Laboratories Ltd., 2019; Klimisch score = 1).

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recognised OECD test guideline and according to Good Laboratory Practice.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
other: Sprague Dawley Crl:CD (SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: ~ 8 to 10 weeks upon arrival
- Weight at study initiation: ~ 225-250 g on GD 3 (day of randomization into groups at RTI)
- Number and sex: 100 timed-mated females; 25 timed-mated (presumed pregnant) females per group and 4 groups
- Mating: Mated at the vendor over 3 days to produce 24, 48, and 28 timed-mated females
- Identification: Microchipped (Bio Medic Data Systems, Seaford, DE) upon arrival at RTI
- Housing: Solid-bottom, polycarbonate caging; Acclimation and gestation: singly
- Enrichment: Nestlets
- Diet (e.g. ad libitum): Purina Certified Rodent Chow No. 5002 (Purina Mills, Inc., Richmond, IN)
Analysis : Provided by supplier Storage: 60-70°F in ARF
Expiration: 6 months after milling date
- Water (e.g. ad libitum): Tap water available ad libitum via an automatic water delivery system (Edstrom Industries, Inc., Waterford, WI)
Source: City of Durham, NC
Analysis reports: Annually by the City of Durham;
Twice yearly by independent laboratory analyses
- Husbandry: All animals used in compliance with the NRC Guide for the Care and Use of Laboratory Animals (2011).
- Bedding: Sani-Chips cage litter (P.J. Murphy Forest Products, Montville, NJ)
- Acclimatation: At least 3 ± 1 days under RTI Animal Research Facility (ARF) conditions. Release by RTI veterinarian for use on study at least 48 hours after arrival.
- Animal Welfare: Humane termination by CO asphyxiation if moribund or evidence of spontaneous abortion or premature delivery. Animals were not subjected to undue pain or distress.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72°F ± 3°F
- Humidity (%): 50 ± 20% RH
- Air changes (per hr): 10-15 per hour, except during scheduled maintenance
- Photoperiod (hrs dark / hrs light): 12-hour light:12-hour dark

IN-LIFE DATES: From: To:
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on analytical verification of doses or concentrations:
A prestudy chemistry was conducted to determine the dietary dose formulations of S-261A in feed.
The doses of 0, 250, 750, and 3750 ppm were accurate (99.2 – 107.2%), homogeneous, and stable for at least 42 days.
The dose formulations were therefore made once and used throughout this study with ad libitum exposure to the maternal animals from GD 6 through scheduled necropsy on GD 20.
Duration of treatment / exposure:
15-day
Frequency of treatment:
Dosed feed or vehicle was available ad libitum 7 days/week from GD 6, with adjustments of feed jars on GD 12 and 18.
Dose / conc.:
0 ppm
Dose / conc.:
250 ppm
Dose / conc.:
750 ppm
Dose / conc.:
3 750 ppm
No. of animals per sex per dose:
Treatment groups: 3
Vehicle control group: 1
Number of animals per group: 25 timed-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
Females were assigned to treatment groups by stratified randomization, by body weight on GD 3, in order to provide uniform mean body weights across dose groups (±20%).
The initial weight range criterion of ±10% was changed to ±20% due to the variance in body weights observed on GD 3 (randomization time). These changes were considered to have no impact on this study.

Treatment groups: 3
Vehicle control group: 1
Number of animals per group: 25 timed-mated females

Dosing Regimen
On GD 6 through 20, S-261A in vehicle or vehicle alone was administered in the feed.
The Study Director and the Sponsor selected the highest concentration (3750 ppm; ~250 mg/kg/day) as one which was expected to induce maternal toxicity or low levels of lethality (< 10%).
The mid dose (750 ppm; ~50 mg/kg/day) was selected based on the expectation of pup developmental and systemic toxicity.
The low dose (250 ppm; ~16.7 mg/kg/day) was anticipated to be a maternal and embryo-fetal NOAEL in this study.
Dosed feed or vehicle was available ad libitum 7 days/week from GD 6, with adjustments of feed jars on GD 12 and 18.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: time the clinical sign was observed
severity (if applicable)
duration (if applicable) of the sign.

Daily morbidity/mortality checks were done twice per day, at least 6 hours apart, beginning the day after receipt.

Maternal observations:
-GD 20 euthanasia by CO2 asphyxiation
-Final body weight (g)
-Organ weights: uterus, liver
-Ovarian corpora lutea counts
-Uterine implantation sites classified as live, dead, early, late, or full resorption
-Dead fetuses (died in utero) recorded as dead and discarded
- Identification and retention of any maternal gross lesions in 10% formalin

Histopathology
Maternal livers were fixed in 10% neutral buffered formalin for subsequent optional histopathology. The formalin-fixed maternal livers from the control (0 ppm) and high dose (3750 ppm) groups were embedded in paraffin and sectioned.
The liver sections were placed on glass slides, stained with hematoxylin and eosin, and coverslipped for histopathologic evaluation.

Ovaries and uterine content:
See Maternal examination box
Fetal examinations:
Euthanasia
Live fetuses were dissected from the uterus and immediately placed on a moist paper towel over a tray of ice. This procedure induces anesthesia and subsequent death by lowering the core body temperature below 25°C (Lumb and Jones, 1973; Blair, 1979).

Fetal Observations:
Fetal Weight and Sex: All live fetuses
External Exam: 100% live fetuses
Fresh Visceral Exam: 50% live fetuses (decapitated)
Fixed Head Examination (Bouin’s): 50% (same fetuses which received visceral examination)
Alizarin Red S/Alcian Blue Skeletal
Staining: 100% (50% intact, 50% decapitated)
Skeletal Evaluation: 50% (intact fetuses)
Storage for Archiving Heads: 70% ethanol
Statistics:
All data were collected on the validated RTI Instem Provantis™ System and software except for fetal external, visceral, and skeletal findings.
For all statistical tests, p< 0.05 was used as the criterion for significance.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Statistically significantly increased absolute and relative liver weights was observed in Group 4 (3750 ppm) maternal females. No evidence of hepatocellular hypertrophy (increase in cell size), or hyperplasia (increase in cell number) were observed in the high dose animals.
Dose descriptor:
NOAEL
Effect level:
750 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
3 750 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
no

Maternal Results

 

Pregnancy: There were 24, 25, 25, and 25 actual timed-pregnant females at 0, 250, 750, and 3750 ppm. 1 in the 0 ppm group was not pregnant.

 

Maternal surviving and abortion: All of the females survived to scheduled termination on GD 20 (no unscheduled deaths) and none of the pregnant females aborted or had a totally resorbed litter.

 

Mean maternal body weights: females were all statistically and biologically equivalent across all groups on GD 3 when they were assigned to groups and on GD 6, 9, 12, 15, 18, and 20 during their gestational exposure period. 

 

Mean maternal body weight gains: Statistically and biologically equivalent on GD 3-6 (prior to start of exposures), 6-9, 9-12, 12-15, 15-18, 18-20, 6-20 (exposure period), and 3-20 (on study).

 

Mean maternal feed consumption in g/animal/day: was equivalent across all groups for all intervals (corresponding to the body weight gain intervals) except for statistically significant increases at 750 ppm (p<0.01) and 3750 ppm (p<0.001) only for GD 12-15. 

 

Mean feed consumption values in g/day:±SEM for that interval were 21.01±0.73 at 0 ppm, 22.40±0.41 at 250 ppm, 23.46±0.48 at 750 ppm, and 24.11±0.58 at 3750 ppm. 

 

Mean maternal feed consumption in g/kg body weight/day for the same gestational intervals was statistically and biologically equivalent across all groups for GD 3-6, 6-9, 9-12, 15-18, and 18-20. However, the maternal feed consumption values were again statistically significantly increased on GD 12-15 for 750 ppm (p<0.01) and 3750 ppm (p<0.001). In addition, maternal feed consumption (g/kg/day) was significantly increased on GD 6-20 at 750 ppm and at 3750 ppm (both at -<0.05) and on GD 3-20 at 750 and 3750 ppm (both at p<0.05).

 

Maternal S-261 intake in mg/kg body weight/day: anticipated increases across groups (with highest values on GD 6-9, lowest values on GD 18-20) for all intervals calculated. For the 3-day intervals, the ranges were 0 mg/kg/day at 0 ppm, 16.775-20.672 mg/kg/day at 250 ppm, 50.539-65.489 mg/kg/day at 750 ppm, and 242.410-314.702 mg/kg/day at 3750 ppm. 

For overall intake on GD 6-20, the mean values were 0.0±0.0, 18.766±0.214, 57.572±0.796, and 287.770±4.016 mg/kg/day at 0, 250, 750, and 3750 ppm, respectively.

 

Maternal clinical observations: 1 female each at 0 and 3750 ppm with scabs, no clinical observations at 250 ppm, and 1 female with alopecia and 1 female with a damaged tail at 750 ppm.

 

Maternal gravid uterine weight, terminal body weight, terminal (GD 20) body weight minus gravid uterine weight, and body weight changes from GD 3-20: all statistically equivalent across all groups.

Maternal liver weights at scheduled necropsy, both absolute and relative to terminal body weights:

-       0, 250, and 750 ppm: equivalent across all groups. 

-       3750 ppm: significantly increased absolute maternal liver weight (by 7.01%) at p<0.01; significantly increased of relative maternal liver weight (by 7.60%) at p<0.001.

 

Histopathology was performed on the high dose and control maternal livers. No maternal hepatic macroscopic (gross) lesions in any livers in any of the groups. No evidence of hepatocellular hypertrophy or hyperplasia in the high dose livers.

 

Note of the pathologist: typically there is no hepatic hypertrophy or hyperplasia until the livers from the treated group(s) are at least 20% heavier than the control livers. In the present study the statistically significant and biologically relevant increases in maternal liver weights were 7.01% (absolute weight) and 7.60% (relative weight) higher than the control livers at the top dose, 3750 ppm. 

It is not known whether the maternal liver weights at the high dose would have increased above the ~7% increases observed, with a longer dosing period.

 

Developmental Toxicity Results

All maternal females had one or more live fetuses at scheduled necropsy on GD 20. 

Number of ovarian corpora lutea, number of uterine implantations per female: equivalent across all groups. 

 

Preimplantation loss/female (number of ovarian corpora lutea minus number of uterine implantation sites), percent preimplantation loss/female: low and equivalent across all groups.

 

No full litter resorptions in any female at any dose was observed. One dead fetus in 1 female at 3750 ppm was recorded.

Incidence of early resorptions and postimplantation loss: low and equivalent across all groups.

 

Mean numbers of live fetuses/litter: equivalent across all groups (11.7, 11.7, 11.4, and 11.4 at 0, 250, 750, and 3750 ppm, respectively;

Percentage of implantations which were live fetuses at term: high and equivalent across all groups (95.56, 97.66, 94.35, and 96.94% at 0, 250, 750, and 3750 ppm, respectively).

 

Alive foetuses/group: 0 ppm: 280; 250 ppm: 292; 750 ppm: 284; 3750 ppm: 285 live

Fetal body weights, expressed as total litter weight, fetal weight per litter (both sexes), and male and female fetal body weight/litter: all equivalent across all groups. 

Percent of male fetuses, relative to the total number of foetuses: equivalent across all groups (51.07, 50.34, 52.46, and 49.47% at 0, 250, 750, and 3750 ppm, respectively.

 

External foetuses malformations and variations.

 

Approximately 50% of each litter was examined for malformations.

No differences across groups for the litter or fetal incidences of external malformations or variations or for incidences of fetal skeletal malformations were observed.

 

There were also no differences across groups for litter or fetal incidences of total malformations or variations, regardless of type (e.g., external, visceral, or skeletal), with 35.7, 38.0, 39.8, and 38.6% of the fetusesin totoexhibiting variations per group and 1.5, 0.7, 3.5, and 2.1% of the fetusesin totoexhibiting malformations per group.

 

There was 1 fetus in 1 litter at 750 ppm with bilateral misshapen limbs, 1 fetus in 1 litter at 3750 ppm with bilateral hydroureter, a different fetus in 1 litter at 3750 ppm with left hydroureter, and 1 fetus in 1 litter each with right hydroureter at 250 and 750 ppm.  

 

Fetal skeletal malformations included: misshapen interparietal bone (3 fetuses in litter at 750 ppm), agenesis of a rib (1 fetus in 1 litter each at 0, 250, 750, and 3750 ppm), discontinuous rib (1 fetus in 1 litter each at 0 and 3750 ppm), fused rib (2 fetuses in 1 litter at 0 ppm), rib cartilage branched (1 fetus in 1 litter at 0 ppm), rib cartilage fused (2 fetuses in 1 litter at 0 ppm), thoracic centrum, bipartite ossification, bipartite cartilage (1 fetus in 1 litter at 750 ppm), and thoracic centrum, dumbbell ossification, and bipartite cartilage (1 fetus in 1 litter at 3750 ppm). 

The remaining fetal findings were classified as variations “not classified” (noted only) and observed in fetuses across all groups.

Conclusions:
A developmental toxicity study was conducted on the test material S-261A, which was administrated to rats at 0, 250, 750, and 3750 ppm. Only at 3750 ppm a maternal hepatic toxicity in the form of increased absolute and relative body weight was observed. Necropsy proved no evidence of hepatocellular hypertrophy or hyperplasia. In addition, no adverse findings were observed in the prenatal offspring at any dose.
Executive summary:

The aim of the study was to investigate the effects ofin uteroexposure to Santicizer 261A (S-261A) on the organogenesis and prenatal development of the CD ®  rat. The test material was administrated to maternal animals for 15-day via the diet during the embryonic and fetal periods (gestational days [GD] 6 through 20). 

The only maternal findings of note were the statistically significantly increased absolute and relative liver weights observed in Group 4 (3750 ppm) maternal females. No evidence of hepatocellular hypertrophy (increase in cell size), or hyperplasia (increase in cell number) were observed in the high dose animals. 

No other treatment- or dose-related maternal findings were observed at any dose or any time during gestation or scheduled necropsy. No effects on maternal body weights, weight gains, feed consumption in g/day or g/kg/day, clinical observations, or pregnancy indices were recorded. No treatment- or dose-related developmental toxicity findings at any dose were observed including no effects on pre- or postimplantation loss, fetal numbers, sex ratio, body weights, or fetal external, visceral, or skeletal malformations or variations. 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The database is of high quality, being based on a study conducted to the recognised OECD TG 414 and under GLP. An additional study, not to a recognised protocol but to GLP, is also avaiable.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The aim of the OECD 414 guideline study was to investigate the effects of in utero exposure to Santicizer 261A (S-261A) on organogenesis and prenatal development of the Sprague-Dawley  rat. The test material was administrated to maternal animals for 15-day via the diet during the embryonic and fetal periods (gestational days [GD] 6 through 20). 

The only maternal findings of note were the statistically significantly increased absolute and relative liver weights observed in Group 4 (3750 ppm) maternal females. No evidence of hepatocellular hypertrophy (increase in cell size), or hyperplasia (increase in cell number) were observed in the high dose animals. 

No other treatment- or dose-related maternal findings were observed at any dose or any time during gestation or scheduled necropsy. No effects on maternal body weights, weight gains, feed consumption in g/day or g/kg/day, clinical observations, or pregnancy indices were recorded. No treatment- or dose-related developmental toxicity findings at any dose were observed including no effects on pre- or postimplantation loss, fetal numbers, sex ratio, body weights, or fetal external, visceral, or skeletal malformations or variations.


Justification for selection of Effect on developmental toxicity: via oral route:
This is a well-conducted study to OECD 414 and GLP

Justification for classification or non-classification

The reproductive toxicity potential of Santicizer 261A (S-261A) has been evaluated in three OECD guideline studies (OECD 414, OECD 416 and OECD 422) as well as a non-standard extended developmental toxicity study design. In all four studies, S-216A was administered to Sprague-Dawley rats via dietary inclusion. In all studies, the predominant systemic toxicity finding was of increased relaltive and absolute liver weight; a finding that is common to the phthalate esters.

In the two-generation study, S-216A was without effect upon any parameter of reproduction up to the highest dietary inclusion level (5000ppm, equivalent to ca. 333-375mg/kg bw/d). Furthermore, S-216A had no effect upon any parameter of prenatal development up to the highest dietary inclusion level (3750ppm, equivalent to ca. 250mg/kg bw/d).

In a modified combined repeated dose toxicity study with the reproduction/developmental toxicity screening test inSprague-Dawley rats (OECD 422),S-216A fed in the diet at up to 6000ppm (ca. 299 mg/kg bw/d in males, 367 mg/kg bw/d in females) was without effect on any fertility or developmental parameters.

In an extended developmental toxicity design, pregnant females were administered diet containing up to 7500ppm S-216A from GD6 through gestation and lactation, and selected F1 male offspring were raised on untreated diet until adulthood. There was a decrease in ano-genital distance in both male and female offspring from all treatment groups compared to control, but not between treatmant groups, and it is questionable as to whether this effect was really related to treatment. Although phthalate esters have been reported to reduce AGD in males, this has not been reported in females. The effect was not solely related to reduced bodyweight, and thus the AGD in the control group offspring are suspected to be abnormally high; no historical control data are given in the report, but comparison of the results from this control group with that from the OECD 416 study does indicate an elevated AGD in both genders in this study. Furthermore, the AGD in F1 males and females in the two-generation study was unaffected by treatment up to 5000ppm in both generations. The report of decreased AGD in males necrposied at PND21 may also therefore be a reflection of this control group value.

Other findings in the F1 males were retained areolae at PND11–13; a slight delay of 1½ days in preputial separation; reduced absolute and relative weight of the LABC at PND21; a slight increase in undescended testes at PND21; and gross pathology changes of the testis. In many cases, these may represent delays in development, since they are resolved with age, presumably with the later surge in testosterone.

Many of these findings were reported at 3750ppm and 7500ppm, but were not observed at the highest dietary inclusion level (5000ppm) in the two-generation study or at the highest dietary inclusion level (6000ppm) in the modified OECD 422 study. Consequently, the relationship of these findings to treatment is not demonstrated or indeed clear, and no classification is proposed.