Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 December 2008 to 23 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was audited by the Wil Quality Assurance Unit throughout the progression of events and determined to have been conducted in compliance with the United States Environmental Protection Agency (EPA) Good Laboratory Practice Standards (40 CFR Parts 160 and 792), 16 October 1989 and 18 September 1989, respectively; the Organisation for Economic Cooperation and Development (OECD) Principles of Good Laboratory Practice [C(97) 186/Final], 26 November 1997; and the Standard Operating Procedures of WIL Research Laboratories, Inc. The study was conducted in accordance with the protocol and protocol amendments as approved by the sponsor and assured by the QA Unit that the final report accurately describes the conduct and findings of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Version / remarks:
OECD Guideline 416;EPA OPPTS 870.3800
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MRD-08-293; JAYFLEX® MB 10
- Physical state: clear, colorless liquid
- Analytical purity: 100%
- Purity test date:
- Lot/batch No.: Lot no. 80317TK110 (MRD-08-293); WIL ID no. 08009D
- Expiration date of the lot/batch: May 2013
- Stability under test conditions: Stable
- Storage condition of test material: Room Temperature
- Other:

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: (P) 7.3 wks; (F1) 13-15 wks
- Weight at study initiation: (P) Males: 214-216 g; Females: 161-162 g; (F1) Males: g; Females: g
- Fasting period before study: N/A
- Housing: Following the initial acclimation period (6days and 3/sex/cage in suspended wire-mesh) all parental F0 and (F1 animals after selection), were housed individually in clean, stainless steel wire-mesh cages suspended above cage-board until pairing. Rats were paired in the home cage of the male. All F0 and F1 parental mated females were transfered to plastic maternity cages with nesting material, ground corncob bedding until weaning on lactation day 21.
- Diet (e.g. ad libitum): Yes
- Water (e.g. ad libitum):Yes
- Acclimation period:14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.7 degrees C to 22.3 degrees C
- Humidity (%):39.3% to 55.3%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12-hour light (0600 hours to 1800 hours) and 12-hour dark


IN-LIFE DATES: From:13Jan09 To:15Sep09

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): prepared approximately weekly
- Mixing appropriate amounts with (Type of food): PMI Nutrition International, LLC Certified Rodent LabDiet 5002
- Storage temperature of food: stored at room temperature in HDPE drums, and considered stable for a maximum
of 10 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): N/A
- Lot/batch no. (if required): N/A
- Purity:N/A
Details on mating procedure:
- M/F ratio per cage:1M:1F
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0 ] of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. N/A
- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged (how): housed in an individual plastic
cage with nesting material.
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyzed dietary formulations (at 91% to102% of target) were within the standard operating procedures range for dietary admixes (85% to 115%) and were homogeneous. Test substance was not detected in the diet offered to the control group (Group 1). Mean Concentration (% w/w) 0.0940, 0.289, and 0.958 for the homogeneity analyses. Analyses were performed with a a high performance liquid chromatography (HPLC) method using ultraviolet (UV) absorbance detection at a wavelength of 229 nm for the determination of MRD-08-293 (MRD-08-157, JAYFLEX® MB10) concentration. The method was validated for analysis of diet admix formulations containing PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 and test article ranging in concentration from 0.1 to 1% (w/w; 1,000 to 10,000 ppm, respectively).
Duration of treatment / exposure:
The control and test diets were offered ad libitum to the F0 and F1 males and females for a
minimum of 70 consecutive days prior to mating. The F0 and F1 males continued to
receive the control and test diets throughout mating and through the day of euthanasia.
The F0 and F1 females continued to receive the control and test diets throughout mating,
gestation, and lactation through the day of euthanasia. The test substance was administered as a constant
concentration (ppm) in the diet.
Frequency of treatment:
F0 males and females were exposed for 127-130 consecutive days and F1 males and females were exposed for
129-144 consecutive days.
Details on study schedule:
- F1 parental animals not mated until 7 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated F1 animals in the study: ~13 weeks (3 weeks PND lactation and 10 weeks dietary feeding).
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Control
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1000 ppm exposure
Basis:
nominal in diet
1000 ppm
Remarks:
Doses / Concentrations:
1000 ppm exposure
Basis:
actual ingested
See table of calculated results below for test substance ingested (Mg/Kg/Day) for F0 and F1, males and females. Intervals addressed for males are prior to mating and after mating. Intervals addressed for females:pre- mating, gestation & lactation.
Remarks:
Doses / Concentrations:
3000 ppm exposure
Basis:
nominal in diet
See table of calculated results below for test substance ingested (Mg/Kg/Day) for F0 and F1, males and females. Intervals addressed for males are prior to mating and after mating. Intervals addressed for females:pre- mating, gestation & lactation.
Remarks:
Doses / Concentrations:
10000 ppm exposure
Basis:
nominal in diet
See table of calculated results below for test substance ingested (Mg/Kg/Day) for F0 and F1, males and females. Intervals addressed for males are prior to mating and after mating. Intervals addressed for females:pre- mating, gestation & lactation.
No. of animals per sex per dose:
30
Control animals:
yes, plain diet
Details on study design:
- Other:
Summary of Calculated F0 GenerationTest Substance Consumption
Mean Calculated F0 Generation Test Substance Consumption Mg/Kg/Day(a)
Males Females
Theoretical
Dietary Prior to After Prior to
Level Mating Mating Mating Gestation Lactation
1000 ppm 71 49 81 65 156
3000 ppm 211 145 249 193 465
10,000 ppm 715 490 790 641 1504

(a) = Summation of mean test substance consumption for the specified interval divided by Number of days or intervals assessed
______________________________________________________________________________________________________
Summary of Calculated F1 Generation Test Substance Consumption
Mean Calculated F1 Generation Test Substance Consumption Mg/Kg/Daya
Males Females
Theoretical
Dietary Prior to After Prior to
Level Mating Mating Mating Gestation Lactation
1000 ppm 92 50 98 64 146
3000 ppm 276 149 292 188 415
10,000 ppm 981 516 1042 640 1434

(a) = Summation of mean test substance consumption for the specified interval divided by Number of days or intervals assessed
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged
labor, delayed labor) or other difficulties.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations:Individual F0 and F1 male body weights were recorded weekly throughout the study and
prior to the scheduled necropsy. Individual F0 and F1 female body weights were recorded weekly until evidence of copulation was observed. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and
20 and on lactation days 1, 4, 7, 14, and 21. Females that had no evidence of mating and for maternal females after weaning (lactation day 21), weekly body weights were recorded until the scheduled 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
- 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): N/A

OTHER:
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of estrus of each F0 and F1 female for 21 days prior to pairing and continued until evidence of mating was observed or until the end of the mating period.
The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P], beginning 21 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of estrus.
Sperm parameters (parental animals):
Parameters examined in all P and F1male parental generations: testis weight, epididymis weight, sperm count in epididymides, sperm motility, sperm morphology. Sperm morphology was evaluated by light microscopy via a modification of the wet-mount evaluation technique (Linder et al., 1992). Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and epididymis from all F0 and F1 males from all test substance-exposed and control groups were weighed, stored frozen, homogenized, and analyzed for determination of homogenization-resistant spermatid count and calculation of sperm production rate (Blazak et al., 1985) using the Hamilton-Thorne IVOS CASA system.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined on PND 1, 4, 7, 14, and 21 in F1 / F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality/survival, presence of gross anomalies, weight gain, physical or behavioural abnormalities (e.g., nursing behavior). Age of attainment of balanopreputial separation was measured in F1 generation pups and age of attainment for vaginal patency was recorded for all F1 females. The anogenital distance of all F2 pups was measured on PND 1. Pups were individually weighed on PND 1, 4, 7, 14, and 21. Mean pup weights were presented by sex for each litter and by group. Pups were individually sexed on PND 0, 4, 14, and 21.

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities; possible cause of death was determined for
pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving F0 adults were euthanized by carbon dioxide inhalation following the selection of the F1 generation and completion of a clinical observation. All surviving F1adults were euthanized by carbon dioxide inhalation following weaning of the F2 pups.
- Maternal animals: All surviving F0 adults were euthanized by carbon dioxide inhalation following the selection of the F1 generation and completion of a clinical observation. All surviving F1adults were euthanized by carbon dioxide inhalation following weaning of the F2 pups.

GROSS NECROPSY
- Gross necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and
spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. females that delivered or had macroscopic evidence
of implantation, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively. The following F0 and F1 parental tissues and organs were collected and were placed in 10% neutral-buffered formalin:
Adrenal glands (2) Lymph node (mesenteric)
Aorta Ovaries [b] and oviducts (2)
Bone with marrow (sternebrae) Pancreas
Brain Peripheral nerve (sciatic)
Cerebrum level 1 Pituitary gland
Cerebrum level 2 Prostate gland
Cerebellum with medulla/pons Salivary gland [mandibular (2)]
Coagulating glands Seminal vesicles (2)
Eyes with optic nerve (2)[a] Skeletal muscle (rectus femoris)
Gastrointestinal tract Skin with mammary gland [c]
Esophagus Spinal cord (cervical)
Stomach Spleen
Duodenum Testes with epididymides (1)[d]
Jejunum and vas deferens
Ileum Thymus gland
Cecum Thyroids [with parathyroids,
Colon if present (2)]
Rectum Trachea
Heart Urinary bladder
Kidneys (2) Uterus with cervix and vagina
Liver (sections of 2 lobes) All gross lesions [e] (all groups)
Lungs (including bronchi,
fixed by inflation with fixative)

[a] = Eyes with optic nerves were fixed in Davidson’s solution.
[b] = Ovaries from all F1 females were transferred to 70% ethanol after 48 hours of fixation in 10% neutral-buffered formalin.
[c] = The mammary gland was collected from females; a corresponding section of skin was taken from the same anatomic area for males.
[d] = The right testis and epididymis were fixed in Bouin’s solution. Both testes and epididymides from animals found dead were fixed in Bouin’s solution.
[e] = Representative sections of corresponding organs from a sufficient number of control group animals were retained for comparison, if possible.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age. Gross necropsy review emphasised developmental morphology and organs of the reproductive system for nonselected F1 and F2 pups euthanized on PND 21.
- These animals were subjected to postmortem examinations (macroscopic and microscopic examination) as follows: Selected F1 and F2 organs (brain, spleen, and thymus) were collected from 1 pups/sex/litter that survived to the scheduled termination on PND 21. These tissues and all gross lesions from F1 and F2 weanlings were preserved in 10% neutral-buffered formalin for possible future histopathologic examination; all other tissues and the carcasses were discarded.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Section 5.8.1.1 were prepared for microscopic examination and weighed, respectively.
Statistics:
All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. The litter was the experimental unit. Mating, fertility, copulation, and conception indices were analyzed using Chi-square test with Yates’ correction factor. Mean parental (weekly, gestation, and lactation) & offspring body weights & body weight changes, parental food consumption, and food efficiency data, estrous cycle lengths, pre-coital intervals, gestation lengths, former implantation sites, live litter sizes, unaccounted-for sites, numbers of pups born, AGD, balanopreputial separation data, vaginal patency data (day of attainment and body weight), absolute and relative organ weights, sperm production rates, epididymal and testicular sperm numbers, and ovarian primordial follicle counts were subjected to parametric one-way analysis of variance (ANOVA) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of postnatal pup survival and pup sexes at birth (percentpercentage of males per litter), percentages of motile and progressively motile sperm, and percentages of sperm with normal morphology were subjected to Kruskal-Wallis nonparametric ANOVA test and Dunn's test for intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used. Two-tailed Fisher’s Exact test was used for histopathology findings.
Reproductive indices:
The following were calculated or measured: Male Mating Index (%), Female Mating Index (%), Male Fertility Index (%), Female Fertility Index (%), Male Copulation Index (%), Female Conception Index (%), Estrous Cycle Length (days), and Pre-Coital Interval (days) for F0 and F1 parental animals.
Offspring viability indices:
Each litter was examined twice daily for survival, and all deaths were recorded. All pups
were individually identified by application of tattoo markings on the digits following
completion of parturition on PND 0. A daily record of litter size was maintained. Intact
offspring dying from PND 0 to 4 were necropsied using a fresh dissection technique,
which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984).
Pups with external abnormalities that would warrant further skeletal examination were
eviscerated and stained (Dawson, 1926) for subsequent skeletal evaluation. Findings
were recorded as either developmental variations (alterations in anatomic structure that
are considered to have no significant biological effect on animal health or body
conformity and/or occur at high incidence, representing slight deviations from normal) or
malformations (those structural anomalies that alter general body conformity, disrupt, or
interfere with normal body function, or may be incompatible with life) as appropriate. A
detailed gross necropsy was performed on any pup dying or euthanized in extremis after
PND 4 and prior to weaning. Euthanasia of pups was performed via an intraperitoneal
injection of sodium pentobarbital prior to PND 11 or via carbon dioxide inhalation on or
after PND 11. Tissues were preserved for possible future histopathological examination
only as deemed necessary by the gross findings.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
F0 - No test substance-related mortality or moribundity was observed for F0 or F1 males and females at any exposure level. No test substance related clinical findings were noted in the F0 or F1 generation at the daily examinations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0 - Lower mean body weight gains were noted in the 10,000 ppm group F0 females generally throughout the entire pre-mating period (study weeks 0-10). No test substance-related effects on mean body weights, body weight gains, or cumulative body weight gains were noted in the 1000 and 3000 ppm group F0 females or the 1000, 3000, and 10,000 ppm group F0 males. Mean body weight gain in the 10,000 ppm group F0 females was significantly (p<0.01) lower than the control group during gestation days 0-4. Mean body weight gains in this group were similar to the control group during gestation days 4-7, 7-11, 11-14, and 14 17. Mean maternal body weights, body weight gains, and cumulative body weight gains in the 1000 and 3000 ppm group F0 females were unaffected by test substance exposure during gestation. Mean body weight in the 10,000 ppm group F0 females was significantly (p<0.01) lower (13.3%) than the control group on lactation day 1, as a result of the lower mean body weight gains noted in this group during the pre-mating and gestation periods. A significant (p<0.01) mean body weight loss (3 g) was noted in this group during lactation days 1-4, compared to a mean body weight gain of 7 g in the control group. Mean maternal body weights, body weight gains, and cumulative body weight gains in the 1000 and 3000 ppm group F0 females were unaffected by test substance exposure during lactation. Differences from the control group were slight and not statistically significant. Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 10,000 ppm group F0 females was generally lower (p<0.05 or p<0.01) than the control group throughout the entire pre mating period (study weeks 0-10) when evaluated on a g/animal/day basis. Following the lactation period (study week 17-18), mean food consumption and food efficiency in the 10,000 ppm group were lower than the control group; differences were generally significant (p<0.01). Mean food consumption and food efficiency in the 1000 and 3000 ppm group F0 females and 1000, 3000, and 10,000 ppm group F0 males were unaffected by test substance exposure.

F1 - Lower mean F1 body weights during the pre-weaning period in the 10,000 ppm group males continued into the post-weaning period. Lower mean body weight gains occurred throughout the pre-mating period (beginning study week 17-18 through study week 27-28); differences from the control group were generally significant (p<0.05 or p<0.01). After the mating period, mean body weight gains in the 10,000 ppm group males were generally similar to the control group; the only significant (p<0.05) difference was a lower mean body weight gain during study weeks 31-32. Mean body weights in this group were significantly (p<0.01) lower compared to the control group throughout the generation (15.7% lower at study week 19 and 12.9% lower by the scheduled termination at study week 36). Due to the decrements in mean body weight gain during the pre-mating period, mean body weight gain was significantly (p<0.01) lower for the entire generation (study weeks 19-36). Mean body weight gains in the 3000 ppm group F1 males were also generally slightly lower than the control group during the pre-mating period. The differences were occasionally significant (p<0.05 or p<0.01). Mean body weight gains in the 3000 ppm group F1 males were also generally slightly lower than the control group during the pre-mating period and the differences were occasionally significant (p<0.05 or p<0.01). The lower mean body weights observed for these F1 females during the pre-weaning period continued into the post-weaning period as mean body weights were generally 10% lower than the control group beginning at study week 19 (after all females had reached PND 22) and continued to be significantly (p<0.01) lower through study week 28 as well as into gestation and lactation. Following the lactation period (study weeks 35 and 36), mean body weights were 9.1% to 10.5% lower than the control group; the differences remained significant (p<0.01).Mean body weight gains for F1 females in the 10,000 ppm group during the pre-weaning period were not observed after weaning; the only significant (p<0.05) difference from the control group was a transient lower mean body weight gain during study week 27-28 (immediately prior to the mating period). Mean body weights in the 10,000 ppm group F1 females were significantly (p<0.01) lower (8.3% to 12.1%) than the control group throughout gestation. No test substance related effects on mean body weights, body weight gains, and cumulative body weight gains were noted in the 1000 ppm group F1 males or the 1000 and 3000 ppm group F1 females.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
F0 - Test substance-related lower mean food consumption was noted in the 10,000 ppm group F0 females during gestation. When evaluated on a g/animal/day basis, differences from the control group were generally significant (p<0.01) and corresponded to a lower mean body weight gain during gestation days 0-4. Mean maternal food consumption and food efficiency in the 1000 and 3000 ppm group F0 females were unaffected by test diet administration during gestation. Differences from the control group were slight and not statistically significant. Mean food consumption, on a g/animal/day basis, was reduced in the 10,000 ppm group F0 females throughout lactation and when the overall lactation period (lactation days 1-21) was evaluated, with the exception of lactation days 4-7, the differences from the control group were significant (p<0.01). Mean maternal food consumption and food efficiency in the 1000 and 3000 ppm group F0 females were unaffected by test diet administration during lactation. Differences from the control group were slight and not statistically significant. Average quantities of MRD-08-293 consumed during the F0 generation are presented below. [In each phase, compared to the low dose the intermediate dose and the high dose were ~3X and ~10X, respectively.] Values for the entire lactation period of all groups were elevated, as is commonly seen in nursing animals when food consumption is increased to support milk production.
Summary of Mean Calculated F0 Generation Test Substance Consumption Mg/Kg/Day(a)
Males Females
Theoretical
Dietary Prior to After Prior to
Level Mating Mating Mating Gestation Lactation
1000 ppm 71 49 81 65 156
3000 ppm 211 145 249 193 465
10,000 ppm 715 490 790 641 1504

(a) = Summation of mean test substance consumption for the specified interval divided by Number of days or intervals assessed
______________________________________________________________________________________________________
F1 - Mean food consumption, evaluated as g/animal/day, was lower in the 10,000 ppm group F1 males and females was lower (generally 2-4 g/animal/day for males and 1-2 g/animal/day when lower for females) throughout the entire generation (males) and pre-mating period (females). The differences from the control group were generally significant (p<0.05 or p<0.01) for males and occasionally significant (p<0.05 or p<0.01) for females. Only the lower food consumption in the males corresponded to test substance-related reductions in mean body weight gains. Mean food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency in the 3000 ppm group F1 males were generally similar to the control group. Test substance-related lower mean maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency were noted in the 10,000 ppm group F1 females compared to the control group during gestation days 0-4. Differences from control were generally significant (p<0.01) and corresponded to lower mean body weight gain during gestation days 0-4. Significantly lower mean food consumption (p<0.01) (2-3 g/animal/day) was also observed during gestation days 4-7, 7-11, and when the entire gestation period (gestation days 0-20) was evaluated. The lower mean food consumption during these intervals did not correspond to reductions in mean body weight gain. Moreover, mean g/kg/day food consumption and food efficiency during these intervals was similar to that in the control group, and significantly (p<0.05 or p<0.01) higher mean food consumption (g/kg/day) and food efficiency were observed in these females during gestation days 14-17. Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency were reduced in the 10,000 ppm group F1 females compared to the control group during lactation days 1-4; the differences were significant (p<0.01 for g/animal/day and food efficiency) and corresponded to mean body weight loss during this interval. Lower mean food consumption (g/animal/day) continued to be noted in the 10,000 ppm group F1 females during the remainder of lactation and when the entire lactation period (lactation days 1-21) was evaluated; the differences were generally significant (p<0.05 or p<0.01). However, during these same intervals, mean food consumption, evaluated as g/kg/day, was similar to the control group and food efficiency was generally significantly higher (p<0.01) than the control group. Mean food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency in the 1000 ppm group F1 males and the 1000 and 3000 ppm group F1 females were unaffected by test substance exposure.
Summary of Mean Calculated F1 Generation Test Substance Consumption Mg/Kg/Day(a)
Males Females
Theoretical
Dietary Prior to After Prior to
Level Mating Mating Mating Gestation Lactation
1000 ppm 92 50 98 64 146
3000 ppm 276 149 292 188 415
10,000 ppm 981 516 1042 640 1434

(a) = Summation of mean test substance consumption for the specified interval divided by Number of days or intervals assessed

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The mean lengths of estrous cycles in the F0 and F1 treated females were similar to the control group values. None of the differences were statistically significant. F0 Estrous Cycle Length (days): 4.2 (Control), 4.0 (Low), 4.2 (Mid), 4.1(high), and Historical Control 4.4 (3.6-5.8). F1 Estrous Cycle Length (days): 4.1(Control), 4.1(Low), 4.1(Mid), 4.2(High) and Historical Control 4.4 (3.6-5.8).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F0 - No test substance-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any exposure level. Differences from the control group were slight and were not statistically significant.
F1 - No test substance related effects were observed on F1 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any exposure level. Mean epididymal sperm concentration in the 1000 and 10,000 ppm groups and left testicular sperm concentration and sperm production rate in the 10,000 ppm group were significantly (p<0.05 or p<0.01) higher than the concurrent control group. However, the concurrent control group values were at the lower end of the historical control values, and increases in these parameters are not considered toxicologically important.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F0 - Results of F0 Reproductive Performance
Exposure Level (ppm) Historical Control
Parameter Control 1000 3000 10,000 Mean (Range)
Male Mating Index (%) 93.3 93.3 96.7 100.0 97.0 (86.7-100.0)
Female Mating Index (%) 93.3 93.3 96.7 100.0 98.4 (86.7-100.0)
Male Fertility Index (%) 90.0 83.3 90.0 100.0 91.2 (73.3-100.0)
Female Fertility Index (%) 90.0 83.3 90.0 100.0 93.3 (73.3-100.0)
Male Copulation Index (%) 96.4 89.3 93.1 100.0 94.5 (78.3-100.0)
Female Conception Index (%) 96.4 89.3 93.1 100.0 95.0 (78.3-100.0)
Estrous Cycle Length (days) 4.2 4.0 4.2 4.1 4.4 (3.6-5.8)
Pre-Coital Interval (days) 2.8 3.0 2.4 3.0 3.0 (1.8-4.8)

F1 - Results of F1 Reproductive Performance
Exposure Level (ppm) Historical Control
Parameter Control 1000 3,000 10000 Mean (Range)
Male Mating Index (%) 96.7 100.0 96.7 96.7 97.0 (86.7-100.0)
Female Mating Index (%) 96.7 100.0 96.7 96.7 98.4 (86.7-100.0)
Male Fertility Index (%) 80.0 90.0 96.7 96.7 91.2 (73.3-100.0)
Female Fertility Index (%) 80.0 90.0 96.7 96.7 93.3 (73.3-100.0)
Male Copulation Index (%) 82.8 90.0 100.0 100.0 94.5 (78.3-100.0)
Female Conception Index (%) 82.8 90.0 100.0 100.0 95.0 (78.3-100.0)
Estrous Cycle Length (days) 4.1 4.1 4.1 4.2 4.4 (3.6-5.8)
Pre-Coital Interval (days) 2.8 2.2 2.8 2.1 3.0 (1.8-4.8)
None statistically significant compared to the control group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
F0 - Test substance-related higher means for F0 male and female relative (to final body weight) liver weights (significant, p<0.01) were observed in the 10,000 ppm group. Histologic changes were not of a type or severity that would be expected to result in an increase in overall liver weight.
F1 - Test substance-related higher means for F1 male and female relative liver weights
(significant, p<0.01) were observed in the 10,000 ppm group. Histologic changes were not of a type or severity that would be expected to result in an increase in overall liver weight. In the F1 females, lower mean absolute pituitary weight in the 10,000 ppm F1 group and higher organ weights (relative to final body weight) for adrenal glands, brain, kidneys, thyroid glands, and thymus weight were treatment related.

GROSS PATHOLOGY (PARENTAL ANIMALS)
F0 - Review of the gross necropsy observations revealed no observations in the F0 males and females that were considered to be associated with administration of the test substance. Macroscopic findings observed in the test substance exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related. No test substance-related effects were observed on the mean numbers of former implantation sites and unaccounted-for sites.
F1 - Review of the gross necropsy observations revealed no observations in the F1 males and females that were considered to be associated with administration of the test substance. No test substance-related effects were observed on the mean numbers of former implantation sites and unaccounted-for sites. The differences between the control and test substance-exposed groups were slight and not statistically significant.

HISTOPATHOLOGY (PARENTAL ANIMALS)
F0 - Periductal fibrosis (minimal to mild) and bile duct hyperplasia (minimal to mild) was observed in the F0 group male livers of the control, 1000, 3000, and 10,000 ppm with minimal (predominently) to mild severity and a higher incidence in test substance-treated group males compared to the control F0 group males. The incidences of periductal fibrosis and bile duct hyperplasia in the F0 animals did not occur in an exposure-related manner, the severity was minimal to mild, the overall background incidence of these findings in the control group was atypically high, and these findings were not observed in a previous subchronic 90-day rat study with this same test substance (Korgaonkar, 2008, WIL-438007). Therefore, these findings in the F0 animals were possibly amplified by the test substance. However, since these changes were of uncertain relationship to the test substance, they are not considered adverse under the conditions of this study.

F1- Periductal fibrosis was observed in the liver of the control, 1000, 3000, and 10,000 ppm F1 group males and females with minimal to mild severity (predominently minimal). Despite an atypically high background incidence of this finding in the F1 male control group, there was an exposure-related increase in the incidence and severity in the 3000 and 10,000 ppm F1 group males and increased incidence in the 1000, 3000, and 10,000 ppm F1 group females compared to the respective F1 control groups. Bile duct hyperplasia was observed in the liver of the control, 1000, 3000, and 10,000 ppm F1 group males with increased incidence and severity in the 3000 and 10,000 F1 group males compared to the F1 control group males. Bile duct hyperplasia was also observed in the 1000, 3000, and
10,000 ppm F1 group females but not in the control F1 group females. Periductal fibrosis and bile duct hyperplasia in the test substance-treated groups occurred at a higher incidence in the F1 generation animals than in the F0 generation; however, the background incidence of these findings was also generally higher in the F1 control group compared to the F0 control group. Therefore, these
findings in the F1 animals were possibly amplified by the test substance, but the changes were of uncertain relationship to the test substance and not considered adverse.

OTHER FINDINGS (PARENTAL ANIMALS)
F1 - The mean number of primordial ovarian follicles in the 10,000 ppm group F1 females was similar to the control group.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 10 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: F0/F1 (migrated information)
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: F0/F1 (migrated information)
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: F0/F1 (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
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
F1 - The mean number of F1 pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0-1, 1- 4 (pre selection), 4 (post-selection) 7, 7- 14, 14- 21, and from birth to PND 4 (pre-selection) and PND 4 (post-selection) to PND 21 were unaffected by the test substance at all exposure levels. Differences from the control group were slight, were not statistically significant, and did not occur in an exposure related manner. The mean number of pups born andlive litter size in the 10,000 ppm group (13.0 and 12.8 pups per dam, respectively) were slightly (not statistically significant) lower than the control group (14.6 and 14.4 pups per dam, respectively); however, the values were within the mean ranges in the historical control data (11.7-16.6 pups per dam and 11.6-16.4 pups per dam, respectively). In addition, the mean numbers of F2 pups born and live litter size in the F2 generation at 10,000 ppm were similar to the control group. Therefore, the lower mean numbers of pups born and live litter sizes in the F1 generation were not considered test substance-related.
F2 - The mean number of F2 pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0-1, 1-4 (pre selection), 4 (post-selection)-7, 7-14, 14-21, and from birth to PND 4 (pre-selection), and PND 4 (post-selection) to PND 21 were unaffected by the test substance at all exposure levels. Slightly lower mean postnatal survival was noted during PND 4 (post selection) to PND 21 in the 3000 and 10,000 ppm groups (95.0% and 93.1% per litter, respectively) compared to the concurrent control group (99.5% per litter). However, because the differences from the concurrent control were of a small magnitude and not statistically significant, and the values were within the historical control data range (86.7% to 100.0% per litter), they were not attributed to test article administration.

CLINICAL SIGNS (OFFSPRING)
F1 - The general physical condition of all F1 pups in this study was unaffected by F0 parental test substance exposure. Pups (litters) that were found dead or euthanized in extremis numbered 15 (11), 11 (8), 16 (8), and 15 (12) in the control, 1000, 3000, and 10,000 ppm groups, respectively. Three (2), 4 (2), 6 (6), and 8 (5) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. Ten pups from 8 litters in the 10,000 ppm group were noted with small stature compared to only 4 pups from 4 litters in the control group; however, this finding was considered secondary to the reduced body weights in this group.
F2 - The general physical condition of all F2 pups in this study was unaffected by F1 parental test substance exposure. Pups (litters) that were found dead numbered 9 (7), 18 (12), 22 (9), and 26 (14) in the control, 1000, 3000, and 10,000 ppm groups, respectively.
Four (3), 8(5), 9(5), and 25(11) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. Although a slight increase in the numbers of pups found dead and/or missing were noted in the 3000 and 10,000 ppm groups compared to the control group, more litters were available for evaluation in these groups and the mean litter proportions of postnatal survival were comparable to the concurrent control group and historical control data.

BODY WEIGHT (OFFSPRING)
F1 - Mean F1 male and female pup birth weights (PND 1) in the 10,000 ppm group were 7.2% and 6.2% lower, respectively, than the control group values; the difference for males was significant (p<0.05). Mean F1 male and female pup body weight gains were significantly (p<0.01) lower than the control group throughout the pre-weaning period (PND 1-21). Mean body weights in the F1 male andfemale pups were significantly (p<0.01) lower (11.2% to 20.6%) than the control group through PND 21. Mean F1 male and female pup body weights and body weight gains in the 1000 and 3000 ppm groups were unaffected by test substance exposure throughout the pre-weaning period. Mean final body weights for F1 males and females in the 10,000 ppm group were significantly (p<0.01) lower (22.4% and 15.2%, respectively) than the control group on PND 21.
F2 - Mean F2 male and female pup birth weights (PND 1) in the 10,000 ppm group were 8.7% and 7.7% lower, respectively, than the controls and differences were significant (p<0.05 or p<0.01). The F2 pup birth weights were ~ 1.5% lower than values for the F1 pup birth weights compared to their respective controls. Mean F2 male and female pup body weight gains were significantly (p<0.05 or p<0.01) lower than the control group throughout the postnatal period. As a result of the low birth weight and decreased body weight gains, mean body weights in the F2 male and female pups were significantly (p<0.01) lower (13.6% to 22.2%) than the control group through PND 21. Mean F2 male and female pup body weights and body weight gains in the 1000 and 3000 ppm groups were unaffected by test substance exposure throughout the postnatal period and no statistically significant differences from the control group were noted.

SEXUAL MATURATION (OFFSPRING)
F1 - A significant (p<0.01) delay in the mean age of attainment of balanopreputial separation was noted in the 10,000 ppm group F1 males (46.2 days) when compared to the control group (44.1 days). Delays (approximately 6 days) in the attainment of balanopreputial separation have been associated with severe decreases (30% to 43%) in mean pup body weights (Carney et al., 2004). Although the pup body weight effect at the age of attainment in the F1 males at 10,000 ppm in the current study was less severe than that reported by Carney et al., the delay in attainment was only approximately 2 days. Test substance exposure at 1000 and 3000 ppm did not affect mean ages of attainment for balanopreputial separation (44.1 and 44.0 days, respectively) or mean body weights (227.5 g and 218.9 g, respectively) when compared to controls (44.1 days and 227.8 g mean body weight). There was no effect on anogenital distance in the F2 pups at 10,000 ppm. Therefore, the delay in attainment of balanopreputial separation in the 10,000 ppm group males was not considered test substance-related.

Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance exposure. Mean ages of attainment of vaginal patency were 34.3, 34.7, and 35.9 days in the 1000, 3000, and 10,000 ppm groups, respectively, when compared to 34.3 days in the control group. Mean body weights at the age of attainment were 124.7 g, 122.9 g, and 110.8 g in the 1000, 3000, and 10,000 ppm groups, respectively, when compared to 120.0 g in the control group. None of the differences from the control group were statistically significant.

ORGAN WEIGHTS (OFFSPRING)
F1 - Mean absolute and relative (to final body weight) spleen and thymus gland weights fo F1 males and females in the 10,000 ppm group were lower than the control group values; differences were generally significant (p<0.05 or p<0.01). Changes were attributed to parental exposure to the test substance.
F2 - Mean final body weights for the F2 males and females in the 10,000 ppm group were significantly (p<0.01) lower (18.4% and 17.4%, respectively) than the control group on PND 21. Mean absolute and relative (to final body weight) spleen and thymus gland weights for F2 males and females in the 10,000 ppm group were lower than the control group values; the differences were generally significant (p<0.05 or p<0.01). Changes were attributed to parental exposure to the test substance.

Mean absolute and relative (to final body weight) spleen and thymus gland weights were higher in the 10,000 ppm group F1 and F2 male and female pups on PND 21. No test substance-related effects on F1 and F2 pup organ weights were observed at 1000 and 3000 ppm.

GROSS PATHOLOGY (OFFSPRING)
F1- Pups (litters) found dead or euthanized in extremis from PND 0 through the selection of the F1 generation numbered 15(11), 11(8), 16(8), and 15(12) in the control, 1000, 3000, and 10,000 ppm groups, respectively. No internal findings that could be attributed to parental exposure to the test substance were noted at the necropsies of pups that were found dead or euthanized in extremis. • No internal findings related to F0 parental exposure to the test diets were observed at the PND 21 necropsy of F1 weanlings selected for organ weights.
F2 - No internal findings attributable to F1 parental test diet exposure were noted at the scheduled necropsy of F2 pups euthanized on PND 21.

HISTOPATHOLOGY (OFFSPRING)


OTHER FINDINGS (OFFSPRING)
The anogenital distances for F2 males (absolute and relative to the cube root of pup body weight) in the 1000, 3000, and 10,000 ppm groups were unaffected by F1 parental test substance exposure. Although the distance relative to the cube root of pup body weight in the 10,000 ppm group F2 males was significantly (p<0.01) higher than the control group males, the difference was attributed to lower pup body weights on PND 1. In addition, a greater anogenital distance for males is not considered adverse.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Pup toxicity no-observed-adverse-effect level for MB 10 when offered in the diet to Crl:CD(SD) rats was considered to be the intermediate exposure level tested.

·        NOAEL = 3,000 ppm for pup toxicity

o       Test article toxicity was evidenced by:

§        Lower birth weights (PND 1) were observed in the F1and F2pups in the 10,000 ppm group and lower mean body weight gains were noted in these pups, resulting in lower mean body weights throughout the pre‑weaning period 

§        The mean age of balanopreputial separation was delayed in the 10,000 ppm group F1males due to low body weight

§        Spleen and thymus gland weights (mean absolute and relative to final body weight) in the 10,000 ppm group F1and F2pups were lower on PND 21

§        Therefore, based on the results of this study, the NOAEL for pup toxicity was considered to be 3000 ppm

Dosage expressed on a body weight basis (average of F0and F1animals), 3000 ppm corresponded approximately to 195 mg/kg/day for males over the entire generation and 271, 191, and 440 mg/kg/day for females during pre-mating, gestation, and lactation, respectively.  

Applicant's summary and conclusion

Conclusions:
Reproductive toxicity no-observed-adverse-effect level for MRD-08-293 (CAS RN 131298-44-7) when offered in the diet to Crl:CD(SD) rats was considered to be the highest exposure level tested.

• NOAEL = 10,000 ppm for reproductive toxicity
o Based on the lack of effects on F0 and F1 reproductive performance: mating, fertility, copulation, and conception indices, estrous cyclicity, and spermatogenic endpoints
o Dosage expressed on a body weight basis (average of F0 and F1 animals), 10,000 ppm corresponded approximately to 676 mg/kg/day for males over the entire generation and 916, 641, and 1469 mg/kg/day for females during pre-mating, gestation, and lactation, respectively.
Parental toxicity no-observed-adverse-effect level for MB 10 when offered in the diet to Crl:CD(SD) rats was considered to be the lowest exposure level tested.

• NOAEL = 1,000 ppm for parental toxicity
o Test article toxicity was evidenced by:
o Lower mean body weights, body weight gains, and food consumption in the 10,000 ppm group F0 and F1 females during the pre-mating, gestation, lactation, and post lactation periods.
o Lower mean body weights, body weight gains, and food consumption were also noted in the 3000 and 10,000 ppm group F1 males.
o Higher mean relative liver weights (associated with lower body weights) were noted in the F0 and F1 parental animals in the 10,000 ppm group.
o Relationship of periductal fibrosis and bile duct hyperplasia to treatment with the test diet (seen in control and treatment levels) was uncertain and the minimal to mild severity of these spontaneous findings was not considered adverse. Findings were of minimal to mild severity and did not occur in a consistent exposure-related manner. The overall background incidence of liver findings was atypically high in the concurrent control groups and the periductal fibrosis and bile duct hyperplasia were not observed in a previous 90-day rat study with Crl:CD(SD) rats from the same source, same test substance, and at the same dosage levels (0, 1000, 3000, and 10000 ppm).
o Therefore, based on the results of this study, the NOAEL for parental toxicity was considered to be 1000 ppm
o Dosage expressed on a body weight basis (average of F0 and F1 animals), 1000 ppm corresponded approximately to 66 mg/kg/day for males over the entire generation and 90, 65, and 151 mg/kg/day for females during pre mating, gestation, and lactation, respectively.
Pup toxicity no-observed-adverse-effect level for MB 10 when offered in the diet to Crl:CD(SD) rats was considered to be the intermediate exposure level tested.

• NOAEL = 3,000 ppm for pup toxicity
o Test article toxicity was evidenced by:
o Lower birth weights (PND 1) were observed in the F1 and F2 pups in the 10,000 ppm group and lower mean body weight gains were noted in these pups, resulting in lower mean body weights throughout the pre weaning period
o The mean age of balanopreputial separation was delayed in the 10,000 ppm group F1 males due to low body weight
o Spleen and thymus gland weights (mean absolute and relative to final body weight) in the 10,000 ppm group F1 and F2 pups were lower on PND 21
o Therefore, based on the results of this study, the NOAEL for pup toxicity was considered to be 3000 ppm
Dosage expressed on a body weight basis (average of F0 and F1 animals), 3000 ppm corresponded approximately to 195 mg/kg/day for males over the entire generation and 271, 191, and 440 mg/kg/day for females during pre-mating, gestation, and lactation, respectively.
Executive summary:

A two-generation study reproduction study was conducted to determine the potential adverse effects of MRD-08-293 (CasRN 131298-44-7) on reproduction. The protocol was designed to be in general accordance with the Organization for Economic Cooperation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 416, Two-Generation Reproduction Toxicity Study, 22 January 2001, and Directive 2004/73/European Community (EC) (O.J. L152 2004) B.35 Two-Generation Reproduction Toxicity Study, and the United States EPA Health Effects Test Guidelines, OPPTS 870.3800, Reproduction and Fertility Effects, August 1998. This included determining the effects of MRD-08-293 on male and female reproductive processes, including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation, and weaning of the F0 and F1 generations and F1 and F2 neonatal survival, growth, and development. One litter per dam was produced in each generation. The offspring of the F0 generation were defined as the F1 litters; pups from the F1 litters were selected to constitute the F1 generation. The offspring of the F1 generation were defined as the F2 litters.

  • Reproductive toxicity no-observed-adverse-effect level for MB 10 when offered in the diet to Crl:CD(SD) rats was considered to be the highest exposure level tested.NOAEL = 10,000 ppm for reproductive toxicity

Based on the lack of effects on F0and F1reproductive performance (mating, fertility, copulation, and conception indices, estrous cyclicity, and spermatogenic endpoints), the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of MRD-08-293 when offered in the diet to Crl:CD(SD) rats was considered to be 10,000 ppm, the highest exposure level tested. When expressed on a body weight basis (average of F0and F1animals), this concentration corresponded approximately 676 mg/kg/day for males over the entire generation and 916, 641, and 1469 mg/kg/day for females during pre-mating, gestation, and lactation, respectively. 

  • Parental toxicity no-observed-adverse-effect level for MB 10 when offered in the diet to Crl:CD(SD) rats was considered to be the lowest exposure level tested.NOAEL = 1,000 ppm for parental toxicity

Parental toxicity was evidenced by lower mean body weights, body weight gains, and food consumption in the 10,000 ppm group F0and F1females during the pre-mating, gestation, lactation, and post-lactation periods. Lower mean body weights, body weight gains, and food consumption were also noted in the 3000 and 10,000 ppm group F1males. Higher mean relative liver weights (associated with lower body weights) were noted in the F0and F1parental animals in the 10,000 ppm group. Periductal fibrosis and bile duct hyperplasia were observed microscopically in the livers of the 1000, 3000, and 10,000 ppm group F0and F1 males and/or females; however, these liver findings of minimal to mild severity did not occur in a consistent exposure-related manner, the overall background incidence was atypically high in the concurrent control groups, and these findings were not observed in a previous 90-day rat study with the same test substance. Therefore, the relationship of periductal fibrosis and bile duct hyperplasia to treatment with the test diet was uncertain and the minimal to mild severity of these findings was not considered adverse. Based on the results of this study, the NOAEL for parental toxicity was considered to be 1000 ppm. When expressed on a body weight basis (average of F0and F1animals), this concentration corresponded approximately 66 mg/kg/day for males over the entire generation and 90, 65, and 151 mg/kg/day for females during pre-mating, gestation, and lactation, respectively.

  • Pup toxicity no-observed-adverse-effect level for MB 10 when offered in the diet to Crl:CD(SD) rats was considered to be the intermediate exposure level tested. NOAEL = 3,000 ppm for pup toxicity. Pup toxicity was evidenced by lower birth weights (PND 1) observed in the F1and F2pups in the 10,000 ppm group. Lower mean body weight gains were noted in these pups, resulting in lower mean body weights throughout the pre-weaning period. The mean age of balanopreputial separation was delayed in the 10,000 ppm group F1males due to low body weight. In addition, mean absolute and relative (to final body weight) spleen and thymus gland weights in the 10,000 ppm group F1and F2pups were lower on PND 21. Therefore, the NOAEL for neonatal toxicity was considered to be 3000 ppm. When expressed on a body weight basis (average of F0and F1animals), this concentration corresponded approximately 195 mg/kg/day for males over the entire generation and 271, 191, and 440 mg/kg/day for females during pre-mating, gestation, and lactation, respectively.

No effects on the following parameters were observed during the study: no effects on anogenital distance, no cryptorchidism, no hypospadias, no effects with regard to testes histopathology and no effects on spermatogenic endpoints. Based on this study, isodecyl benzoate does not have any endocrine disrupting properties.