Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzyl, C7-C9 linear and branched alkyl esters, 1,2 benzene
dicarboxylic acid
- Lot/batch No.: 1K3039
- Storage condition of test material: Tightly closed container in a cool, well-ventilated area

Test animals

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.

Administration / exposure

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.
Doses / concentrations
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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

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)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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

Details on results (F1)

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.

Effect levels (F1)

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)

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.