Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Mar 2021 to 31 Aug 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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)

Version / remarks:

OECD Guidelines for the Testing of Chemicals, Section 4, Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

EC Number:

915-318-3

Cas Number:

41199-19-3, 17283-81-7 and 31499-72-6

IUPAC Name:

Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

Test material form:

liquid

Details on test material:

Identification: POLYAMBROL® (also known as 1DF139-POLYAMBROL®)
Batch/Lot No.: T007930
Retest Date: 11 Sep 2023
Physical Description: Colorless liquid
Purity: 100%
Correction Factor: N/A
Storage Conditions (temperature set to maintain): Controlled room temperature

Specific details on test material used for the study:

No further details specified in the study report

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

At this time, 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.
The Sprague Dawley rat was selected because: 1) it is a standard species accepted for use in fertility and early embryonic development studies; 2) this species and strain has been demonstrated to be sensitive to reproductive toxicants; and 3) historical data and experience exist at the Testing Facility.
The number of animals chosen for this study is the smallest number considered necessary to provide the minimum number of pregnancies recommended by the applicable guidelines. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Ninety (90) virgin (43) male and (47) female Crl:CD(SD) Sprague Dawley rats were received in filtered cartons by truck from the Charles River Laboratories, Inc. facility located in Raleigh, NC.
The body weight range for the male rats was 393 g to 484 g on the day of dose administration. The female rats were 87 days of age upon arrival at the Testing Facility.
The body weight range for the female rats was 222 g to 271 g on the day of dose administration. The female rats were 80 days of age upon arrival at the Testing Facility.

Animal Identification
P Generation: A subcutaneously implanted electronic identification chip.
F1 Generation: On Day 0 postpartum, all F1 generation pups were individually identified by paw tattoo prior to anogenital distance measurements using Testing Facility Standard Operating Procedures (SOPs).

Environmental Acclimation
Method: After receipt at the Testing Facility, the rats were acclimated for at least 6 days prior to initiation of estrous cycling (female rats) or initiation of exposure (male rats).

Selection, Assignment, Replacement, and Disposition of Animals
Disposition: The disposition of all animals was documented in the study records.
Selection and Assignment : P Generation
Before initiation of exposure, males were selected for study on the basis of physical condition and body weights recorded during acclimation. Females were selected for study on the basis of physical condition, body weights recorded during the acclimation period and evidence of normal estrous cycling. The male rats were assigned to dose groups, based on computer-generated (weight-ordered) randomization procedures.
Eighty (80) rats were assigned to 4 groups (Groups 1 through 4), 10 rats/sex/group.

Husbandry
Housing
Housing: During the acclimation and estrous evaluation period, the animals were socially housed (2 to 3 per box), when possible.
During the exposure period, the animals were individually housed, except during the cohabitation and postpartum periods. During the cohabitation period, one male rat and one female rat were pair housed in the male rat’s solid-bottomed cage. After cohabitation, animals were returned to individual housing. Each dam and delivered litter were housed in a common nesting box during the postpartum period.
Caging: Polycarbonate cages containing appropriate bedding.
Housing set-up was as described in the Guide for the Care and Use of Laboratory Animals. Carrier control group animals were housed on a separate rack from the test substance-exposed animals.

Environmental Conditions
The conditions for animal room environment were as follows:
Temperature: Targeted Range: 66°F to 77°F (19°C to 25°C)
Actual Range: 67°F to 78°F
Humidity: Targeted Range: 30% to 70%
Actual Range: 29% to 68%
Light Cycle: 12 hours light and 12 hours dark (except during designated procedures)
Ventilation: At least 10 changes per hour of fresh air that has been passed through 99.97% HEPA filters.

Animal Enrichment
Type/Frequency: Animals were provided with Crink l’Nest™, a resting platform, and a chewing item, except when interrupted by study procedures/activities.
Analysis: There were no known contaminants in the animal enrichment that would interfere with the objectives of the study.

Bedding
Type: Bed-o'Cobs®
Frequency: Changed as often as necessary to keep the animals dry and clean.
Analysis: Results of analysis for environmental contaminants are on file at the Testing Facility. There were no known contaminants in the bedding that would interfere with the objectives of the study.

Food
Diet: During the acclimation period, animals were provided Certified Rodent Diet® #5002 meal (PMI® Nutrition International).
During the exposure period, animals were provided the same diet only (Group 1) or diets prepared using the same diet and the test substance (Groups 2 through 4).
On the day prior to euthanasia, 5 rats/sex/group were fasted (no more than 18 hours) prior to scheduled euthanasia for the purpose of clinical pathology sample collection.
Type: Meal
Frequency: Ad libitum, except during designated procedures.
Analysis: Results of analysis for nutritional components and environmental contaminants are on file at the Testing Facility. There were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Type: All water was from a local source and passed through a reverse osmosis membrane before use. Chlorine was added to the processed water as a bacteriostat; processed water was expected to contain no more than 1.2 ppm chlorine at the time of analysis.
Frequency/Ration: Available ad libitum.
Analysis: Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility. There were no known contaminants in the water that could interfere with the objectives of the study.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The diets were prepared at least weekly, and all prepared batches were assigned an 8-day shelf life. The prepared diet batches were stored in a refrigerator set to maintain -20°C when not in use. The dispensed feed jars were maintained at ambient conditions in the animal room for up to 8 days from the date of preparation.
Detailed preparation procedures (Formulation Batch Records [FBR]) were maintained in the raw data. Any residual amounts were discarded.

Details on mating procedure:

Frequency: Maximum of 14 days.
Within each exposure group, consecutive order was used to assign rats to cohabitation (i.e., pairing, one male per one female). The cohabitation period consisted of a maximum of 14 days. Females with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be GD 0 and assigned to individual housing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses was performed by HPLC-UV using a validated analytical procedure (Validation No. 1557.RTD1.01).

Concentration and Homogeneity Analysis
Sample Allocation: Duplicate for analysis, triplicate for backup.
Storage Conditions: Temperature set to maintain -20°C.
Acceptance Criteria: For concentration: mean sample concentration results within or equal to ± 15% of theoretical concentration. Each individual sample concentration result within or equal to ± 20%.
For homogeneity, relative standard deviation (RSD) of concentrations of ≤10% for each group.

Stability Analysis
Stability analyses performed previously in conjunction with Study No. 01557004 demonstrated that the test substance is stable in the carrier control substance when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Study No. 01557004.
Additional 4-day room temperature stability is being performed concurrently with this study. The criteria for this additional stability data was ± 10% from the time 0 results.

Duration of treatment / exposure:

Males: Fourteen (14) days before cohabitation, during cohabitation and continuing through the day before euthanasia. Males were exposed to the test substance and/or the carrier control substance for a minimum of 43 days.
Females: Fourteen (14) days before cohabitation, during cohabitation and continuing until Lactation Day 13.

Frequency of treatment:

Continuous exposure.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 000 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

5 000 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

2 500 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

0 ppm (nominal)

No. of animals per sex per dose:

10 per sex/per dose group

Control animals:

yes, plain diet

Positive control:

Not required for the study

Examinations

Parental animals: Observations and examinations:

Viability • At least twice daily. -

Clinical Observations: General
• At least once weekly during the acclimation period.
• Daily during the exposure period.
• On the day of scheduled euthanasia.

Clinical Observations: Detailed: Once before the start of the exposure period (baseline) and once weekly thereafter. During the week of FOB testing, detailed clinical observations will not be conducted on the 5 rats/sex/group receiving FOB evaluations. Detailed clinical observations were conducted for all male and female rats outside the cage in a standard arena at the same time each day of conduct. Effort was made to ensure that variations in the test conditions are minimal and that observations are conducted by observers unaware of treatment groups. Signs noted should include, but not be limited to changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behavior (e.g., self-mutilation, walking backwards) were recorded.

Individual Body Weights
Male Rats
• On the day after arrival.
• Twice weekly during the acclimation period.
• Twice weekly during the exposure period and on the day of scheduled euthanasia.
Female Rats
• Twice weekly during the exposure period.
• On GDs 0, 3, 7, 10, 14, 17, 21 and 25, if necessary.
• LD 0, 4, 7, 10, 13 and 13. -

Food Consumption
Male Rats
• At least twice weekly during the exposure period until cohabitation.
• At least twice weekly after all male rats have mated.
Female Rats
• At least twice weekly during the exposure period until cohabitation.
• On GDs 0, 3, 7, 10, 14, 17, 21 and 25, if necessary.
• LD 0, 4, 7, 10, and 13. During cohabitation, when two rats occupied the same nesting box with one food jar, replenishment of the food jars was documented. Individual values were not recorded or tabulated.

Functional Observational Battery (FOB)
Procedure: The FOB was conducted by an observer unaware of the group assignment of the rat. The observer examined the rat in its home cage, while handling the animal, and/or in an open field to assess parameters including, but not limited to the following:
• Home Cage Observations
• Posture/body carriage, convulsions, stereotypy, tremors, palpebral closure/ptosis
• Handling Observations
• Ease of removal, handling reactivity
• Open Field Observations
• Rearing, arousal/alertness, gait/mobility, vocalizations, tremor, respiration, defecation, urination, stereotypy, convulsions, appearance, lacrimation, salivation, exophthalmos, palpebral closure/ptosis, erected fur
• Sensorimotor Observations
• Touch response/tactile reflex, auditory startle response, pain response (via tail pinch method), pupil response
• Body Temperature
• Neuromuscular Observations
• Body tone (via extensor thrust reflex), grip strength, air righting reflex

Motor Activity
Procedure: The rats were placed in an individual enclosure held within a Smartframe containing 7 x 15 photobeams utilizing infra-red pyroelectric detectors. Movement was detected in 2 dimensions anywhere in the enclosure and was differentiated into fine movement and ambulation. Each animal was monitored for one session of 60 minutes.
For the purpose of data tabulation, activity data files were reduced to Excel® format into successive periods of 10 minutes each at the completion of testing. Fine movements and ambulation were analyzed in these six 10-minute periods and compared across the exposure groups.

Clinical Pathology
Sample Collection
On the day of scheduled euthanasia, blood was collected from 5 rats/sex/group from P generation rats assigned to the study as outlined in table form, see "Any other information for details".. Blood was collected via the vena cava while under isoflurane/oxygen anesthesia from rats assigned to the study. Rats were euthanized via exsanguination, following blood sample collection. Animals were fasted on the night before scheduled euthanasia (no longer than 18 hours; water was available ad libitum). The time of blood collection was documented in the raw data.
Correspondence regarding clinical pathology was sent to the Principal Investigator. Samples were shipped to Charles River Laboratories Ashland, LLC, Ashland, OH.

Hematology
All samples were placed on a tilter and maintained at ambient conditions. All samples were checked for clots. Samples that contain clots were not shipped or analyzed.
Blood samples were stored refrigerated until shipment. Differential leukocyte slides were maintained and shipped at ambient conditions.

Coagulation
Blood samples were processed for plasma, and plasma was analyzed for the parameters listed in
Blood was transferred into 1.0 mL sodium citrate tubes and centrifuged within 30 minutes of collection at room temperature for at least 15 minutes. The resulting plasma samples was frozen on dry ice as soon as possible until stored in a freezer set to maintain -60°C until shipment for analysis.

Clinical Chemistry
Blood samples were processed for serum, and the serum was analyzed for the parameters specified in Table for, see "Any other information" for details.

Thyroid Hormone Analysis
P Generation
On the day of scheduled euthanasia, blood samples were collected from all P generation male and female rats via the inferior vena cava while under isoflurane/oxygen anesthesia. All P generation samples were collected in the morning (between 8 and 11 am) Schedule detailed in table form, see "Any other information for details".

Sample Processing
Blood samples were placed into serum separator tubes, allowed to clot for at least 20 minutes on ice (not to exceed 1 hour) and centrifuged at room temperature at 3500 rpm for 15 minutes. The resulting serum was separated, transferred to uniquely labeled clear polypropylene tubes, and frozen immediately over dry ice or in a freezer set to maintain 70°C.
Samples retained from the P generation male and the F1 generation pups (Day 13 postpartum only) were shipped for analysis. All other samples (P generation females and F1 generation culled pups) were retained at the Testing Facility for future possible evaluation.
The serum samples were shipped (frozen on dry ice) to Charles River Laboratories, Ashland, LLC, Ashland, OH.

Sample Analysis
Serum samples were analyzed for Thyroxine (T4) by Charles River Ashland Bioanalytical Chemistry using a validated UHPLC/MC/MS assay (Lucarell JM, 2018, 00099764) and according to Charles River SOPs, and the results will be included in the Final Report.

Oestrous cyclicity (parental animals):

• 14 consecutive days before initiation of exposure.
• 14 consecutive days beginning with the day after the first day of exposure then until spermatozoa was observed in a smear of the vaginal contents and/or a copulatory plug is observed in situ during the cohabitation period.
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.

Sperm parameters (parental animals):

Not specified in the study report.

Litter observations:

Viability: At least twice daily. Counted at least once daily (pups in each litter). -
Clinical Observations: General: At least once daily starting on Day 0 postpartum. -
Individual Body Weights: Day 0, 4, 7, 10, 13 postpartum. -
Anogenital Distance: PND 0/1 Males and Females
Nipple/Areolae Retention : PND 13 Males

Thyroid Hormone Analysis
On Day 4 postpartum, blood samples were collected from 2 culled pups/litter/group via decapitation. Note: If there is only one pup available above the culling target, only one pup was culled and used for blood collection for possible serum T4 assessments. Samples collected on Day 4 postpartum were pooled by litter.
On Day 13 postpartum, blood samples were collected from 1 pup/sex/litter/group via decapitation.
All F1 generation samples were collected in the morning (between 8 and 11 am).

Postmortem examinations (parental animals):

Method of Euthanasia
P rats assigned to the study were euthanized via exsanguination following blood collection under isoflurane/oxygen anesthesia.

Scheduled Euthanasia
P generation male rats were euthanized approximately 2 weeks following the completion of the cohabitation period.

Ovarian and Uterine Examinations
The reproductive tract was dissected from the abdominal cavity. The number of implantation sites was recorded.

Necropsy
A gross necropsy of the thoracic, abdominal and pelvic viscera was performed for each animal.

Organ Weights
The organs identified for weighing were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, unless otherwise specified. Organ to body weight ratio (using the terminal body weight) and organ weight to brain weight ratio were calculated for the P generation.

Tissue Collection and Preservation
Representative samples of the tissues were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated. A table of random units was used to select one P generation carrier control group rat per sex from which all tissues examined at necropsy was retained, in order to provide carrier control tissues for any possible future evaluations of gross lesions.

Histology and Histopathology
Histology
Tissues were processed at the Charles River Laboratories International, Inc. facility located in Frederick, MD. Tissues from the first 5 consecutive animals/sex from Groups 1 and 4 P generation, gross lesions from Groups 1 to 4 P generation, thyroid/parathyroid glands from remaining 5, Group 1 and 4 P generation and all Groups 2 and 3 P generation identified in the Terminal Procedures table were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Histopathology
Histopathological evaluation was performed by a board-certified veterinary pathologist on the following:
• Thyroid and parathyroid was evaluated from all P generation males and females in all groups.
• Gross lesions were evaluated in all P generation males and females in all groups.
• Special attention was paid to the stages of spermatogenesis in the testes, epididymides, and interstitial testicular cell structures.
Target tissues identified by the study pathologist during microscopic evaluation will be communicated to the Study Director; tissues will be evaluated and reported.
At the discretion of the study pathologist and after acknowledgement by the Study Director, images may be captured for consultation purposes.

Postmortem examinations (offspring):

Reported in table form, see "Any other information" for details.

Statistics:

Any data collected during the pre-exposure period was not tabulated, summarized, or statistically analyzed, unless applicable to summarization in the following sections. All statistical analyses were performed within the respective study phase, unless otherwise noted.
Clinical and necropsy observations data were summarized but no inferential statistical analysis were performed.
Numerical data collected on scheduled occasions was summarized and statistically analyzed as indicated below according to sex and occasion or by litter.

Reproductive indices:

Number of Estrous Cycles and Mean Cycle Length
Pregnancy, Mating and Fertility Indices
Pre-coital Interval

Offspring viability indices:

Natural Delivery and Litter Observations (Proportional) (e.g. Pregnant, Females with Liveborn, Gestation Index, Female with Liveborn)
Natural Delivery and Litter Observations (Count) (e.g. Gestation Length, Live Pups, Implantation Sites)
Litter Observations (Continuous) (e.g. Sex Ratio - Males, Mean Litter Bodyweights)
Live Birth Index
Developmental Landmark (Cumulative %) Anogenital Distance
Developmental Landmark (Cumulative %) Nipple/areola retention

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Male Rats
All clinical signs that were observed were considered unrelated to the test substance because: 1) the observations were not dose-dependent; and/or 2) the observations occurred in only one rat in a dose group and the clinical signs did not persist. These clinical signs included vocalization; bent tail; erected fur; thin fur cover, discolored skin; a scab on the skin; hypersensitive; hyperreactive; and increased activity.

P Generation-Fenale Rats
All clinical signs that were observed were considered unrelated to the test substance because: 1) the observations were not dose-dependent; and/or 2) the observations occurred in only one rat in a dose group and the clinical signs did not persist. These clinical signs included hunched posture; erected fur; hypersensitive; abnormal respiratory rate; thin fur cover, a scab on the skin; increased activity; and abnormal consistency of the feces.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All male rats survived until scheduled euthanasia.
All female rats survived until scheduled euthanasia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male rats
There were no adverse changes in body weight or body weight gain observed in the male rats during the exposure period. At the end of the exposure period (on DS 36), the mean body weights in the male rats were 99%, 97% and 97% of the control group value in the 2500, 5000 and 10000 ppm exposure groups, respectively. There was a statistically significant decrease (p≤0.01) in the mean body weight on DS 4 at 10000 ppm in comparison with the control group value. At 5000 ppm, there was a decrease in body weight gain observed on DSs 1 to 4 in comparison with the control group value. A statistically significant body weight loss (p≤0.01) was observed at 10000 ppm on DSs 1 to 4 in comparison with the control group value. These differences at the initiation of the exposure period were considered to be related to taste aversion to the diet and were not considered to be adverse. There was a statistically significant increase (p≤0.05) in body weight gain observed at 10000 ppm on DSs 4 to 8 in comparison with the control group value. This increase was considered to be a recovery from the initial body weight loss observed on DSs 1 to 4 and was not considered to be adverse. On DSs 25 to 29, there were statistically significant decreases (p≤0.05) in body weight gain observed at 5000 and 10000 ppm in comparison with the control group value. These changes in body weight gain were not considered to be test substance related because they were single occurrences and did not persist.

P Generation-Female Rats
At 5000 and 10000 ppm, there were statistically significant decreases (p≤0.05 or p≤0.01) in body weight gains during the premating period (DSs 1 to 15) in comparison with the control group value. On DSs 1 to 4, there were statistically significant decreases (p≤0.05 or p≤0.01) in body weight gains at 2500 and 5000 ppm and a statistically significant (p≤0.01) body weight loss observed at 10000 ppm in comparison with the control group value.
During the gestation phase, there were statistically significant decreases (p≤0.05) in mean body weights at 5000 ppm on DGs 7, 14 and 17 in comparison with the control group values. At 10000 ppm, there were statistically significant decreases (p≤0.01) in mean body weights at all intervals between DGs 3 to 17 in comparison with the control group values. Body weight gains were also decreased or statistically significantly decreased (p≤0.05 or p≤0.01) at 2500, 5000 and 10000 ppm on DGs 0 to 3 and at 5000 and 10000 ppm on DGs 3 to 7 in comparison with the control group values.
During the lactation phase, there were decreases or statistically significant decreases (p≤0.05 or p≤0.01) in mean body weights at 10000 ppm at all intervals between DL 0 and DL 10 in comparison with the control group values.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male Rats
At 5000 and 10000 ppm, there were statistically significant reductions (p≤0.05 or p≤0.01) in food consumption on DSs 1 to 4 in comparison with the control group value.
Food consumption was unaffected by exposure to the test substances in the males at all intervals prior to cohabitation at 2500 ppm.

P Generation-Female Rats
During the premating phase, there were statistically significant decreases (p≤0.01) in food consumption observed at 2500, 5000 and 10000 ppm during the premating period (DSs 1 to 15) in comparison with the control group value. There were also statistically significant decreases (p≤0.05 or p≤0.01) in food consumption observed on DSs 1 to 4 and 11 to 15 at 5000 ppm and at all intervals at 10000 ppm in comparison with the control group values.
During the gestation phase, there were decreases or statistically significant decreases (p≤0.05 or p≤0.01) in food consumption observed on DGs 0 to 3, 3 to 7 and 7 to 10 at 2500, 5000 and 10000 ppm in comparison with the control group values. There was also a statistically significant decrease (p≤0.01) in food consumption observed at 10000 ppm on DGs 14 to 17 in comparison with the control group value.
There were also statistically significant decreases (p≤0.05) in food consumption at 10000 ppm on DLs 7 to 10 and 10 to 13 in comparison with the control group values.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Male Rats
There were no test substance-related differences in hematology and coagulation values at exposure levels up to and including 10000 ppm.
Any differences in hematology and coagulation values, regardless of statistical significance, were consistent with biological variation and were considered unrelated to exposure to the test substance.

P Generation-Female Rats
There were no test substance-related differences in hematology and coagulation values at exposure levels up to and including 10000 ppm.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Male Rats
There were no test substance-related differences in clinical chemistry values at exposure levels up to and including 10000 ppm.
Any differences in clinical chemistry values, regardless of statistical significance, were consistent with biological variation and were considered unrelated to exposure to the test substance.

P Generation-Female Rats
There were no test substance-related differences in clinical chemistry values at exposure levels up to and including 10000 ppm.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no test substance-related effects on the Functional Observational Battery evaluation in the male rats at exposure levels up to and including 10000 ppm.
There were no test substance-related effect on the motor activity evaluation in the male rats at exposure levels up to and including 10000 ppm.
There were no test substance-related effects on the Functional Observational Battery evaluation in the female rats at exposure levels up to and including 10000 ppm.
There were no test substance-related effect on the motor activity evaluation in the female rats at exposure levels up to and including 10000 ppm.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Male Rats
In the kidneys of males at ≥ 2500 ppm, POLYAMBROL®-related findings included an increased incidence and/or severity of chronic progressive nephropathy (CPN) compared to controls as well as accumulation of hyaline droplets in the tubules of the renal cortex and degeneration/necrosis of tubules, predominately in the region of the corticomedullary junction. These findings are consistent with those reported in the literature where many chemicals have been found to exacerbate the incidence and/or severity of CPN and that hyaline droplet nephropathy was usually associated with a concomitant increase in the severity of CPN. Since the findings in these males were not dose-dependent, there were no increases in blood urea nitrogen or creatinine in the serum clinical chemistry, and the relevance to humans are uncertain7, the findings were not considered adverse.
No POLYAMBROL®-related microscopic findings were noted in the testes and the testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to exposure to POLYAMBROL®.

P Generation-Female Rats
In the five P generation females examined, findings in the reproductive organs including aggregates of macrophages, often pigmented, in the wall of the uterus, mucification of the vaginal epithelium, mature corpora lutea in the ovaries and mammary gland hyperplasia with secretory product were consistent with the animals having been pregnant and lactating and, therefore, were considered normal.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to exposure to POLYAMBROL®.

Other effects:

no effects observed

Description (incidence and severity):

Natural Delivery and Litter Observations
Mating occurred in 10, 10, 9 and 10 females and pregnancy occurred in 10, 10, 9 and 10 females at 0, 2500, 500 and 10000 ppm, respectively.
No test substance-related effects were noted on any natural delivery or ovarian and litter observation parameters in the P generation females. The values for the duration of gestation, the gestation index, females completing delivery, females with liveborn pups, the mean number of live newborn pups, the percentage of the live birth index and the percentage of the post implantation loss per litter were similar among the four exposure groups. There were statistically significant decreases (p≤0.05 or p≤0.01) in the total implantation sites observed at 2500, 5000 and 10000 ppm in comparison with the control group values. These decreases were not considered to be adverse as there were no differences observed in the number of live newborn pups observed in these exposure groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All estrous cycling parameters were unaffected by exposure to the test substance at doses as high as 10000 ppm.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Male Rats
All mating and fertility parameters were unaffected by exposure to the test substance at levels up to and including 10000 ppm.
Female Rats
All mating and fertility parameters were unaffected by exposure to the test substance at doses as high as 10000 ppm.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

10 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

2

Key result

Dose descriptor:

NOAEL

Effect level:

10 000 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical observations in the F1 generation pups were attributable to maternal exposure to the test substance at doses as high as 10000 ppm.
All clinical signs that were observed were considered unrelated to the test substance because: 1) the observations were not dose dependent; and/or 2) the observations occurred in only one pup in a particular dose group. These clinical signs included discolored skin; a scab on the skin; and abdominal distension.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Pup body weights were unaffected by maternal exposure to the test substance at doses up to and including 10000 ppm. At 10000 ppm, there were statistically significant decreases (p≤0.05 or p≤0.01) in the mean pup body weights on PNDs 10 and 13 in comparison with the control group values. This decrease in pup body weight is considered to be related to the pups starting to consume the diet and being exposed directly to 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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances for the male and female pups were comparable across all exposure groups and there was no statistically significant difference.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no male pups observed with the presence of nipples on study.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No POLYAMBROL®-related thyroid gland weight changes were noted in the F1 generation pups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related gross findings were noted in the F1 generation male and female pups. The gross findings observed were considered incidental and, therefore, were considered unrelated to exposure to POLYAMBROL®.

Histopathological findings:

no effects observed

Description (incidence and severity):

No POLYAMBROL®-related microscopic findings were noted in the thyroid/parathyroid glands in the F1 generation pups.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

10 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

10 000 ppm (nominal)

Treatment related:

no

Any other information on results incl. tables

Male Rats

Consumed Doses

	Target dose level (ppm)
	2500	5000	10000
DSs 1 to 15
Mean Calculated Dose (mg/kg/day)	153.959	300.235	572.937
Maximum Weekly Dose (DSs)	158.011 (4 to 8)	315.679 (4 to 8)	654.980 (4 to 8)
DS = Day of study.

 

Males Rats

Summary of Male Microscopic Findings – Terminal Euthanasia (P Generation)

Group	1	2	3	4
Dose (ppm)	0	2500	5000	10000
No. Animals Examined	10	10	10	10
Kidney (No. Examined)	10	10	10	10
Chronic Progressive Nephropathy	(5)^a	(8)	(10)	(9)
Minimal	5	5	5	4
Mild	0	3	4	5
Moderate	0	0	1	0
Accumulation, hyaline droplets, tubular	(0)	(10)	(10)	(10)
Mild	0	4	3	2
Moderate	0	6	6	8
Marked	0	0	1	0
Degeneration/necrosis, tubular	(0)	(4)	(6)	(4)
Minimal	0	4	5	4
Mild	0	0	1	0
^a  Numbers in parentheses represent the number of animals with the finding.

 

P Generation-Female Rats

Consumed Doses

	Target dose level (ppm)
	2500	5000	10000
DSs 1 to 15
Mean Calculated Dose (mg/kg/day)	166.688	315.399	568.682
Maximum Dose (DSs)	174.645 (4 to 8)	341.408 (4 to 8)	656.363 (11 to 15)
DGs 0 to 17
Mean Calculated Dose (mg/kg/day)	181.490	355.265	672.249
Maximum Dose (DGs)	186.190 (14 to 17)	374.226 (14 to 17)	691.656 (14 to 17)
DLs 0 to 10
Mean Calculated Dose (mg/kg/day)	352.215	649.204	1396.544
Maximum Dose (DLs)	426.746 (7 to 10)	780.204 (7 to 10)	1561.612 (7 to 10)
DS = Day pf study; DG = Day of gestation; DL = Day of lactation.

 

P Generation-Female Rats

Summary of Female Microscopic Findings – Terminal Euthanasia (P Generation)

Group	1	2	3	4
Dose (ppm)	0	2500	5000	10000
No. Animals Examined	10	10	10	10
Kidney (No. Examined)	5	0	0	5
Chronic Progressive Nephropathy	(0)	(-)	(-)	(0)
Minimal	0	-	-	0
Mild	0	-	-	0
Moderate	0	-	-	0
Accumulation, hyaline droplets, tubular	(0)	(-)	(-)	(0)
Mild	0	-	-	0
Moderate	0	-	-	0
Marked	0	-	-	0
Degeneration/necrosis, tubular	(0)	(-)	(-)	(0)
Minimal	0	-	-	0
Mild	0	-	-	0
^a  Numbers in parentheses represent the number of animals with the finding.

 

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for general toxicity was 10000 ppm in the male rats and was not established in the female rats. In the females, reductions in body weight gain or a body weight loss and reduced food consumption were observed at all doses during the premating and gestation phase. Reductions in mean body weights were observed at 5000 and 10000 ppm during the gestation phase and at 10000 ppm during the lactation phase.
The reproductive NOAEL was 10000 ppm as there were no test substance-related changes in estrous cycling in the female and mating and fertility in the males or females. The NOAEL for viability and growth in the offspring was also 10000 ppm.

Executive summary:

The objectives of this study were to test for toxic effects/disturbances resulting from repeated exposure to Crl:CD(SD) male and female rats to POLYAMBROL® in the diet beginning before cohabitation, through mating and continuing for 49 days (male rats) or through parturition until Lactation Day 13 (female rats).  This study was designed to incorporate a reproduction/developmental toxicity screening test that is used to provide initial information on possible effects on male and female reproductive performance (e.g., gonadal function, mating behavior, conception, development of the conceptus and parturition).  The study also places emphasis on neurological effects as a specific endpoint and identifies the neurotoxic potential of a test substance, which may warrant further in-depth investigation.

The study design was as follows:

 

Experimental Design

Group No.	Test Material	Test Substance (ppm)	No. of Animals
			Males	Females
1	Carrier Control Substance	0	10	10
2	POLYAMBROL^®	2500	10	10
3	POLYAMBROL^®	5000	10	10
4	POLYAMBROL^®	10000	10	10
ppm = Parts per million.

 

The following parameters and end points were evaluated in this study: viability, clinical observations, body weights and body weight gains, food consumption, estrous cycle evaluations, cohabitation and mating, Functional Observational Battery (FOB), Motor Activity, clinical pathology parameters (hematology, coagulation, and clinical chemistry), thyroid hormone analysis, anogenital distance, nipple/areolae retention, organ weights, and histology and histopathology examinations.

Male Rats

The mean calculated doses for the male rats during the exposure period were 153.959, 300.235 and 572.937 in the 2500, 5000 and 10000 ppm exposure groups respectively.

All male rats survived until scheduled euthanasia.

There were no test substance-related clinical signs observed in the male rats.

There were no adverse changes in body weight, body weight gain or food consumption observed in the male rats during the exposure period.

There were no test substance-related effects on the Functional Observational Battery or motor activity evaluations in the male rats at exposure levels up to and including 10000 ppm.

All mating and fertility parameters in the male rats were unaffected by exposure to the test substance at levels up to and including 10000 ppm.

There were no test substance-related differences in hematology, coagulation and clinical chemistry values in the male rats at exposure levels up to and including 10000 ppm.

No test article-related gross findings were noted in the male rats.  

No test substance-related organ weight changes were noted in the male rats.  

In the kidneys of males at ≥ 2500 ppm, POLYAMBROL®-related findings included an increased incidence and/or severity of chronic progressive nephropathy (CPN) as well as accumulation of hyaline droplets in the tubules of the renal cortex and degeneration/necrosis of tubules, predominately in the region of the corticomedullary junction.  These findings are consistent with those reported in the literature where many chemicals have been found to exacerbate the incidence and/or severity of CPN and that hyaline droplet nephropathy was usually associated with a concomitant increase in the severity of CPN.  Since the findings in these males were not dose-dependent, there were no increases in blood urea nitrogen or creatinine in the serum clinical chemistry, and the relevance to humans are uncertain, the findings were not considered adverse.  

Examination of the testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis.

Female Rats and F1 Generation

The mean calculated doses for the female rats during the premating period were 166.688, 315.399 and 568.682 in the 2500, 5000 and 10000 ppm exposure groups respectively. The mean calculated doses for the female rats during the gestation period were 181.490, 355.265 and 672.249 in the 2500, 5000 and 10000 ppm exposure groups respectively.  The mean calculated doses for the female rats during the lactation period were 352.215, 649.204 and 1396.544 in the 2500, 5000 and 10000 ppm exposure groups respectively.

All female rats survived until scheduled euthanasia.

There were no test substance-related clinical signs observed in the female rats.

During the premating phase, there were statistically significant decreases in body weight gains at 5000 and 10000 ppm during the premating period (DSs 1 to 15).  There were also statistically significant decreases in food consumption observed at 2500, 5000 and 10000 ppm during the premating period.  On DSs 1 to 4, there were statistically significant decreases in body weight gains at 2500 and 5000 ppm and a statistically significant body weight loss observed at 10000 ppm.  There were also statistically significant decreases in food consumption on DSs 1 to 4 and 11 to 15 at 5000 ppm and at all intervals at 10000 ppm. 

During the gestation phase, there were statistically significant decreases in mean body weights at 5000 ppm on DGs 7, 14 and 17 in comparison with the control group values.  At 10000 ppm, there were statistically significant decreases in mean body weights at all intervals between DGs 3 to 17.  Body weight gains were also decreased or statistically significantly decreased at 2500, 5000 and 10000 ppm on DGs 0 to 3 and at 5000 and 10000 ppm on DGs 3 to 7.  There were decreases or statistically significant decreases in food consumption observed on DGs 0 to 3, 3 to 7 and 7 to 10 at 2500, 5000 and 10000 ppm.  There was also a statistically significant decrease in food consumption observed at 10000 ppm on DGs 14 to 17.

During the lactation phase, there were decreases or statistically significant decreases in mean body weights at 10000 ppm at all intervals between DL 0 and DL 10 and statistically significant decreases in food consumption at 10000 ppm on DLs 7 to 10 and 10 to 13.

All estrous cycling, mating and fertility parameters were unaffected by exposure to the test substance at exposure levels as high as 10000 ppm.

There were no test substance-related effects on the Functional Observational Battery and motor activity evaluations in the female rats at exposure levels up to and including 10000 ppm.

Mating occurred in 10, 10, 9 and 10 females and pregnancy occurred in 10, 10, 9 and 10 females at 0, 2500, 500 and 10000 ppm, respectively.

No test substance-related effects were noted on any natural delivery or ovarian and litter observation parameters in the P generation females. 

No clinical observations in the F1 generation pups were attributable to maternal exposure to the test substance at exposure levels as high as 10000 ppm.

Pup body weights were unaffected by maternal exposure to the test substance at doses up to and including 10000 ppm.  

The anogenital distances for the male and female pups were comparable across all exposure groups and there was no statistically significant difference.  There were no male pups observed with the presence of nipples on study.

There were no test substance-related differences in the hematology, coagulation or clinical chemistry values at exposure levels up to and including 10000 ppm.

There were no test substance-related gross findings, organ weight changes or any adverse histopathology in the P generation or F1 generation rats.  

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for general toxicity was 10000 ppm in the male rats and was not established in the female rats.  In the females, reductions in body weight gain or a body weight loss and reduced food consumption were observed at all doses during the premating and gestation phase.  Reductions in mean body weights were observed at 5000 and 10000 ppm during the gestation phase and at 10000 ppm during the lactation phase. 

The reproductive NOAEL was 10000 ppm as there were no test substance-related changes in estrous cycling in the female and mating and fertility in the males or females.  The NOAEL for viability and growth in the offspring was also 10000 ppm.