2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct. 2021 - Sept 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Currently, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
This species and strain of rat is recognized as appropriate for reproduction studies. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The number of animals is based on the OECD Guideline for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 July 2016.

Test material

Test material information

Constituent 1

Reference substance name:

2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated

EC Number:

500-022-5

EC Name:

2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated

Cas Number:

9014-85-1

Molecular formula:

variable

IUPAC Name:

2,4,7,9-Tetramethyl-5-decyne-4,7-diol, ethoxylated

Constituent 2

Reference substance name:

2,4,7,9-Tetramethyl-5-decyne-4,7-diol , ethoxylated

IUPAC Name:

2,4,7,9-Tetramethyl-5-decyne-4,7-diol , ethoxylated

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 to 11 weeks
- Weight at study initiation: 225 to 450 g (males)/175 to 300 g (females)
- Housing:
Group housed (2–3 animals of the same sex and same dosing group together) during the premating period.
During cohabitation, the females were paired (1:1) with a male for mating (home cage of the male).
Single/Individual following positive signs of mating or the end of the breeding period.
In Solid-bottom cages containing appropriate bedding material (Bed-OCobs ® or other suitable material). For enrichment, animals were provided items such as treats, a gnawing device, nd/or nesting material, except when interrupted by study procedures/activities.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water (e.g. ad libitum): Municipal tap water, treated by reverse osmosis and ultraviolet irradiation, ad libitum
- Acclimation period: a minimum of 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 26°C
- Humidity (%): 30% to 70%
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark.

IN-LIFE DATES:
From: 12 Oct. To: 21. Dec. 2021

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: diet; PMI Nutrition International, LLC LabDiet Certified Rodent Diet 5002

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): PMI Nutrition International, LLC LabDiet Certified Rodent Diet 5002
- Storage temperature of food: -20°C

Details on mating procedure:

Following a minimum of 14 days of exposure, 1 female was cohabited with 1 male rat of the same treatment group, avoiding sibling mating, in a solidbottom cage for mating (home cage of the male). A maximum of 14 days was allowed for mating. If no evidence of mating is obtained after 14 days, the animals were separated without further opportunity for mating, and the female was placed in a solidbottom cage containing bedding material.

Analytical verification of doses or concentrations:

yes

Remarks:

by gas chromatography (GC) with flame ionization detection (FID)

Details on analytical verification of doses or concentrations:

Diet formulation samples were collected for analysis for analysis of homogeneity and concentration.
Dose analysis results were verified prior to diet administration at each sampling interval, if
possible. If results are deemed unacceptable, the diet formulations will be prepared again and
analyzed.

Duration of treatment / exposure:

Males: for at least 14 days prior to mating and continuing throughout
mating until euthanasia for a minimum of 28 days.

Females: for at least 14 days prior to mating and continuing throughout
mating, gestation and lactation until euthanasia.

Frequency of treatment:

continuously

Details on study schedule:

at least 7 days of acclimatisation
minimum of 14 days pre mating exposure
minimum of 14 days mating period
males. termination of 28 days of exposure
females: exposure during gestation and lactation
culling/first homone analysis of pups: LD 4
temination of females and pups on LD 14

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

800 ppm

Remarks:

in diet (corresponding to 58/68 mg/kg bw/d for males/females); the test substance was administered as a constant concentration (ppm) in the diet with the following exceptions: The concentration of test substance in the diet was adjusted to 0.5X for females that deliver, during Lactation Day (LD) 4 through euthanasia, to account for approximately 2X higher maternal food consumption (and consequently higher test substance consumption) which is normally associated with
the higher demands of milk production.

Doses / concentrations 3

Dose / conc.:

2 500 ppm

Remarks:

in diet (corresponding to 184/220 mg/kg bw/d for males/females); the test substance was administered as a constant concentration (ppm) in the diet with the following exceptions: The concentration of test substance in the diet was adjusted to 0.5X for females that deliver, during Lactation Day (LD) 4 through euthanasia, to account for approximately 2X higher maternal food consumption (and consequently higher test substance consumption) which is normally associated with
the higher demands of milk production.

Doses / concentrations 4

Dose / conc.:

7 500 ppm

Remarks:

in diet (corresponding to 539/668 mg/kg bw/d for males/females); the test substance was administered as a constant concentration (ppm) in the diet with the following exceptions: The concentration of test substance in the diet was adjusted to 0.5X for females that deliver, during Lactation Day (LD) 4 through euthanasia, to account for approximately 2X higher maternal food consumption (and consequently higher test substance consumption) which is normally associated with
the higher demands of milk production.

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on dose range finding study
- Fasting period before blood sampling for clinical biochemistry: overnight
- Post-exposure recovery period in satellite groups: no
- Dose range finding studies: 14 d oral toxicity (diet)

Examinations

Parental animals: Observations and examinations:

- Mortality: twice daily
- Detailed clinical observations: weekly, beginning on the first day of test diet administration for males through euthanasia, and beginning at the start of estrous lavages for females, until evidence of copulation is observed, or until euthanasia (for females with no evidence of mating).
Gestation Days 0, 4, 7, 11, 14, 17, and 20.
Lactation Days 1, 4, 7, 10, and 13.
Mortality and all signs of overt toxicity will be recorded on the day observed. The observations shall include, but are not limited to, evaluations for changes in appearance of skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous systems, somatomotor activity, and behavior.
- Cage-side observations: daily
- Individual body weight:
F0 males: weekly
F0 females: Weekly beginning with the first day of test diet administration until evidence of copulation is observed, or until euthanasia (for females with no evidence of mating).
Gestation Days 0, 4, 7, 11, 14, 17, and 20.
Lactation Days 1, 4, 7, 10, and 13.
- Food consumption:
F0 males: daily
F0 females: Daily, beginning with the first day of test diet administration until the initiation of breeding.
Gestation Days 0-20 (daily).
Lactation Days 1-13 (daily).
Estrous: Daily vaginal lavages performed beginning during the pretest period and continuing through the 2-week premating and mating treatment periods until evidence of mating is observed. Lavaging was continued for those females with no evidence of mating until termination of the mating period.
- Breeding: detection of mating
- Parturition, lactation and littering: Females were observed for dystocia (prolonged or difficult labor) or other difficulties and any findings were recorded. When parturition is judged to be complete, the sex of each pup was determined, pups were examined for gross malformations, and the number of stillbirths and live pups were recorded. Any changes or abnormalities in nesting and nursing behavior were recorded.
- FOB:
males: during the last week (days 24-28); 5/group
females: lactation day 13; 5/group
Home cage observations (Biting, Convulsions/tremors, Feces consistency, Palpebral (eyelid) closure, Posture), handling observations (Ease of handling animal in hand, Ease of removal from cage, Eye prominence, Fur appearance, Lacrimation/chromodacryorrhea, Mucous membranes/eye/ skin color, Muscle tone, Palpebral closure, Piloerection, Red/crusty deposits, Respiratory rate/character, Salivation), open field observations (Arousal, Backing, Bizarre stereotypic behavior, Convulsions/tremors, Gait, Gait score, Grooming, Mobility, Rearing, Time to first step (seconds), Urination/defecation), sensory observations (Air righting reflex, Approach response, Eye blink response, Forelimb extension, Hind limb extension, Olfactory orientation, Pupil response, Startle response, Tail pinch response, Touch response), neuromuscular observations (Grip strength-hind and forelimb, Hind limb extensor strength, Hind limb foot splay, Rotarod performance), physiological observations (Body temperature, Body weight, Catalepsy)
- Motor activity:
males: during the last week (days 24-28); 5/group
females: lactation day 13; 5/group
Motor activity was measured using the Kinder Scientific Motor Monitor System (Kinder Scientific Company, LLC, Chula Vista, CA). Locomotor activity sessions was performed in a room equipped with a white noise generator set to operate at 70 ± 10 dB.

Oestrous cyclicity (parental animals):

Daily vaginal lavages performed beginning during the pretest period and continuing through the 2-week premating and mating treatment periods until evidence of mating is observed. Lavaging was continued for those females with no evidence of mating until termination of the mating period.

Litter observations:

- Mortality: daily
- Detailed clinical observations: PND 1, 4, 7, 10 and 13. Animals were removed from the cage. Mortality and all signs of overt toxicity were recorded on the day observed. The observations shall include, but are not limited to, evaluations for changes in appearance of skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous systems, somatomotor activity, and behavior Any abnormalities in nesting and nursing behavior were recorded.
- Cage-side observations: daily
- Pup sex: PND 0 or 1, 4 and 13
- Individual pup body weights: PND 1, 4, 7, 10 and 13
- Anogenital distance: PND 1
- Assessment of areolae/nipple retention: PND 13

Postmortem examinations (parental animals):

- Thyroid hormone assessment (T3/T4):
F0: all males at termination and all females on LD 13

- Clinical pathology:
F0 males: on day of scheduled necropsy
F0 females: LD 14

Hematology: Red blood cell count, Hemoglobin concentration, Hematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular hemoglobin concentration, Mean corpuscular, hemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells (absolute), Other cells (as appropriate), Mean platelet volume
Coagulation: Activated partial thromboplastin time, Fibrinogen, Prothrombin time
Clinical chemistry: Alanine aminotransferase, Albumin, Albumin/globulin ratio, Alkaline phosphatase, Aspartate aminotransferase, Bile acids, Calcium, Chloride, Creatinine, Gamma-glutamyltransferase, Globulin (calculated), Glucose, Phosphorus, Potassium, Sodium, Sorbital dehydrogenas, Total bilirubin, Cholesterol, Total protein, Triglycerides, Urea nitrogen

- Necropsy: all F0 animals (all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. For parental females, the number of former implantation sites were recorded).

- Organ weights: all F0 animals; brain, epididymis, pituitary, prostate, seminal vesicle, thyroid, heart, kidney, liver, ovary, spleen, testis, thymus

- Microscopy: all F0 animals (control and high dose group; mid and low dose group in case of observed lesions); aorta, bone marrow (sternum), brain, epidymis, esophagus, eye, adrenal, coagulating gland, pituitary, prostate, salivary gland (mandibular), seminal vesicle, thyroid (incl. parathyroids), heart, kidney, cecum, colon, rectum, liver, lung, lymph nodes (axillary, mandibular, mesenteric), skeletal muscle, macroscopic abnormalities, optic/sciatic nerve, ovary, oviduct, pancreas, peyer's patches, skin, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testis, thymus, trachea, urinary bladder, uterus/cervix, vagina, vas defens

Postmortem examinations (offspring):

- Thyroid hormone assessment (T3/T4):
two pups/litter at PND 4 and one pup/sex/litter at PND 13

- Alizarin Red S staining: in case of suspected skeletal anomlaies
- Gross necopsy: pups older than 5 days (all pups found dead/euthanized in extremis, 1 pup/sex/litter on PND 13)
- preservation of thyroid (incl. parathyroids): 1 pup/sex/litter

Statistics:

All analyses were two-tailed for significance levels of 5% and 1%. All means were presented
with standard deviations.
in-life data:
Continuous data variables (body weights, body weight changes, and food consumption at each interval), estrous cycle length, pre-coital intervals, gestation length, former implantation sites, unaccounted-for sites, thyroid hormone values, anogenital distance (absolute and relative to cube root of body weight), number of nipples/areolae (only when nipples are present), clinical pathology values, and Functional Observational Battery data values will be analyzed by a parametric one-way analysis of variance (ANOVA). If the results of the ANOVA are significant (p<0.05), Dunnett’s test will be applied to the data to compare the treated groups to the control group. Functional Observational Battery parameters which yield scalar or descriptive data will be analyzed by Fisher’s Exact test.
Male copulation, female conception, and male and female mating and fertility indices of the treated groups will be compared to the control group using the Chi-square test with Yates’ correction factor.
Litter data:
The mean litter proportions (% per litter) of pup viability during the postnatal period and sex ratio at birth will be subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference. If the results of the ANOVA are significant (p<0.05), Dunn’s test will be applied to the data to compare the treated groups to the control group. Mean litter weights, live litter size, and number of pups born will be subjected to the parametric ANOVA test and Dunnett’s test as described above with the litter representing the experimental unit.
Organ weight data:
Organ weights (absolute and relative to body weights and relative to brain weights) will be subjected to a parametric ANOVA test and Dunnett’s test as described above.
Locomotor activity data:
Two-sided test.

Reproductive indices:

mating index, fertility index, copulation index, female conception index, pre-coital interval

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical observations were noted at the detailed examinations or cage-side observations for males and females at any dietary concentration.

Mortality:

no mortality observed

Description (incidence):

All F0 males and females survived to the scheduled necropsy.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean body weights and body weight gains in the 800, 2500, and 7500 ppm group animals were unaffected by test substance exposure throughout the study.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on hematology parameters in the 800, 2500, and 7500 ppm group males and females when evaluated on Study Day 29 or Lactation Day 14, respectively.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes in clinical chemistry were noted for males and females at all dietary concentrations. In the 800, 2500, and 7500 ppm groups, lower mean total bilirubin concentrations were noted for males and females compared to the control group; differences were statistically significant for males at 2500 and 7500 ppm and for females at 7500 ppm.
Additionally, statistically significantly higher mean albumin, total protein, and globulin concentrations were noted for males in the 7500 ppm group compared to the control group. These differences were not considered adverse due to the lack of corresponding adverse microscopic findings.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on thyroid hormone values in the F0 males at any dietary concentration.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage parameters were unaffected by test substance exposure. Handling, open field, neuromuscular and sensory parameters as well as motor activity patterns were unaffected by test substance exposure. There were no statistically significant differences for the test substance-exposure groups when compared to the control group on Study Day 28 (males) or on Lactation Day 13 (females).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minimal to mild hepatocellular hypertrophy noted in 2500 and 7500 ppm group males and 7500 ppm group females was characterized by increased cytoplasm and increased nuclear diameter. The microscopic distribution of this finding was diffuse in males and centrilobular in females.
Thyroid follicular hypertrophy noted in 2500 and 7500 ppm group males was characterized by increased follicular cell height, increased cytoplasm, and reduced colloid content.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean lengths of estrous cycles in these groups were similar to the control group value.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on reproductive performance (mating index, fertility index, copulation index, female conception index, pre-coital interval) were observed at any dietary concentration.

Details on results (P0)

- Gestation Length and Parturition
Mean gestation lengths in the 800, 2500, and 7500 ppm groups were unaffected by test substance exposure. No statistically significant differences were noted. No signs of dystocia were noted in these groups.
- Litter data
The mean number of pups born, live litter size and the percentage of males at birth in 800, 2500, and 7500 ppm groups were similar to the control group values

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

2 500 mg/kg bw/day

Based on:

test mat.

Remarks:

corresponding to 180 and 220 mg/kg bw/d mean substance intake for males and females, respectively

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

7 500 ppm

Based on:

test mat.

Remarks:

corresponding to 539 and 668 mg/kg bw/d mean substance intake for males and females, respectively

Sex:

male/female

Basis for effect level:

other: no effects on reproductive performance observed

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance exposure.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Postnatal survival in all groups was unaffected by test substance exposure.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weights and body weight changes during PND 1–13 in the 800, 2500, and 7500 ppm groups were unaffected by parental exposure of the test substance.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on thyroid hormone values in the F1 males and females at any dietary concentration on PND 13.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 800, 2500, and 7500 ppm groups were similar to the control group values

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was unaffected by parental exposure to the test substance when evaluated on PND 13. No retained nipples/areolae were noted for any male pup.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No internal findings were noted at the necropsy of pups euthanized on PND 13.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

7 500 ppm

Based on:

test mat.

Remarks:

corresponding to 539 and 668 mg/kg bw/d mean substance intake for males and females, respectively

Sex:

male/female

Basis for effect level:

other: no developmental effects observed

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Please refer to attachment

Applicant's summary and conclusion

Conclusions:

Based on the lack of effects on F0 reproductive parameters and F1 neonatal survival and growth, a dietary concentration of 7500 ppm (the highest concentration tested) was considered to be the NOAEC for F0 reproductive toxicity, F0 neurotoxicity, and F1 neonatal toxicity. This concentration corresponded to mean dose levels of 539 and 668 mg/kg/day for F0 males and females during the premating period, Gestation Days 0-20, Lactation Days 1–4, and Lactation Days 4–13, respectively.

Executive summary:

In the present OECD 422 study animals were exposed to the test substance continuously via the diet at concentration of 0, 800, 2500 and 7500 ppm. F0 males were exposed for 14 days prior to mating and continuing until euthanasia for a total of 28 days. F0 females were exposed for 14 days prior to mating and continuing through Lactation Day 13. F1 offspring were potentially exposed to the test substance in utero and via maternal milk, as well as via direct consumption of the diet during the latter portion of the lactation period.

Dietary concentrations of the test substance were adjusted to 0.5x for females that delivered, during Lactation Day 4 through euthanasia, to compensate for the approximately 2x higher maternal food consumption (and consequently higher test substance consumption) that is normally associated with the higher caloric demands of milk production.

The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, neurobehavior, thyroid hormones, clinical pathology (hematology and clinical chemistry), organ weights, and macroscopic and microscopic examinations.

 

Mean calculated test substance consumption:

Males:

58 (800 ppm), 184 (2500 ppm) and 539 (7500 ppm) mg/kg bw/d

Females:

- prior to mating: 52 (800 ppm), 170 (2500 ppm) and 513 (7500 ppm) mg/kg bw/d

- Gestation: 59 (800 ppm), 184 (2500 ppm) and 548 (7500 ppm) mg/kg bw/d

- Lactation days 1 - 4: 94 (800 ppm), 324 (2500 ppm) and 941 (7500 ppm) mg/kg bw/d

- Lactation days 4 - 13: 66 (800 ppm), 200 (2500 ppm) and 669 (7500 ppm) mg/kg bw/d

 

All F0 males and females survived to the scheduled necropsy. No test substance-related clinical observations were noted at the detailed examinations or cage-side observations for males and females at any dietary concentration.

Lower mean food consumption was noted for F0 males and females in the 7500 ppm group during Study Days 0–1 compared to the control group and was considered test substance-related but not adverse due to the transient nature of the changes and based on the lack of corresponding effects on body weight. No other test substance-related effects on body weights, body weight gains, food consumption, and food efficiency were noted for F0 males and females at any dietary concentration throughout the exposure period.

There were no test substance-related effects on FOB parameters (home cage observations, handling observations, open field observations, sensory observations, neuromuscular observations, and physiological observations) or locomotor activity parameters for F0 males when evaluated on Study Day 28 or for F0 females when evaluated on Lactation Day 13 at any dietary concentration.

Higher mean albumin, total protein, and globulin levels were noted in the 7500 ppm group males when compared to the control group. Lower mean total bilirubin levels than the controls were noted in the 800, 2500, and 7500 ppm group males and females; differences were statistically significant in the 2500 ppm group males and the 7500 ppm group males and females. These findings were considered test substance-related but not adverse due to the lack of adverse corresponding microscopic findings. No test substance-related effects on hematology parameters were noted for F0 males and females.

No test substance-related effects on T4 concentrations were noted for F0 males at any dietary concentration.

Exposure by dietary administration resulted in test substance-related, higher liver weights in the 2500 ppm group females and the 7500 ppm group males and females, higher thyroid/parathyroid weights in the 7500 ppm group males, nonadverse hepatocellular hypertrophy and thyroid follicular hypertrophy in the 2500 and 7500 ppm group males, and nonadverse hepatocellular hypertrophy in 7500 ppm group females. The effects on liver weights in the 7500 ppm group males and females were considered adverse due to the magnitude of changes. There were no test substance-related macroscopic observations.

 

The mean number of pups born, live litter size and the percentage of males at birth in 800, 2500, and 7500 ppm groups were similar to the control group values. Postnatal survival in these same groups was unaffected by test substance exposure. The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance exposure. Mean male and female pup body weights and body weight changes during PND 1–13 in the 800, 2500, and 7500 ppm groups were unaffected by parental exposure of the test substance. The anogenital distances (absolute and relative to the cube root of pup body weight) in the 800, 2500, and 7500 ppm groups were similar to the control group values. Areolae/nipple anlagen in the F1 male pups was unaffected by parental exposure to the test substance when evaluated on PND 13. No retained nipples/areolae were noted for any male pup. There were no test substance-related effects on thyroid hormone values in the F1 males and females at any dietary concentration on PND 13. No internal findings were noted at the necropsy of pups euthanized on PND 13.