Registration Dossier

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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 November 2021 - 17 March 2023
This information will be submitted later based on ECHA decission number TPE/CCH-F-2114356598-33-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
There is a final decision for this study based on ECHA decission number TPE/CCH-F-2114356598-33-01. Interm report provided as completion of the study was not obtained by the requested deadline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2023

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
Study based on ECHA decission number TPE/CCH-F-2114356598-33-01.

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
EC Number:
276-014-8
EC Name:
Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
Cas Number:
71786-60-2
Molecular formula:
Not applicable
IUPAC Name:
2,2'-(C12-18 evennumbered alkyl imino) diethanol
Test material form:
liquid
Details on test material:
- Chemical name: Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
- CAS number: 276-014-8

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Strain RccHan®:WIST. (Han Wistar)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals

Species: Rat.
Strain: RccHan®:WIST. (Han Wistar)
Age: ordered Males: 71 to 77 days of age. Females: 64 to 70 days of age.
Weight range: ordered Males: 300-349 g. Females: 165-195 g.
Number of animals ordered: 108 males and 108 females (216 in total); males and females must not be siblings. (100 F0 animals per sex allocated to study; 8 F0 animals per sex spare).
Supplier: Males: Envigo RMS (Netherlands). Females: Envigo RMS (UK).

Acclimatization

Duration: At least 5 days.
Age at start of treatment: Males: approximately 11 to 12 weeks of age. Females: approximately 10 to 11 weeks of age.
Age at start of pairing: Males: approximately 13 to 14 weeks of age. Females: approximately 12 to 13 weeks of age.
Husbandry conditions Refer to Section 6.2.

Allocation to Treatment Groups (F0 Generation)

Allocation: After a period of acclimatization.
Method: By sex. After exclusion of animals showing signs of illhealth. Animals at the extremes of the body weight range will not be selected if alternatives are available.
At commencement of the study the weight variation should not exceed 20% of the mean weight of each sex.
Cage distribution: Arrangement designed to minimize environmental variables.

Selection of Offspring to form F1 Generation

Selection: On Day 18-20 of age.
Allocation: At weaning on Day 21 of age
Formal start of F1generation: Nominally Day 28 of age (± 2 days of age)
Number per group: See section 6.3.1.
Method: Where possible, two male and two female offspring will be selected from each selected litter for F1 Cohorts 1A and 1B (up to the required number of offspring). For F1 Cohorts 2A and 2B at least 1 male or 1 female offspring will be selected from each selected litter (up to the number required). Where possible 1 male or female will be selected from the litters used to populate Cohort 2B. Additionally, at least 1 male or 1 female offspring will be selected to make up Cohort 1C in order to enable the evaluation of sexual maturation of up to 60 male and 60 female offspring.
Selected animals will be microchipped on Day 18-20 of age and separated from littermates on Day 21 of age.
Up to two male and two female F1 offspring per group will be retained as spares, to provide potential replacement in the event of any mortalities. These spares will have body weights and clinical signs monitored weekly and will be terminated after commencement of the F1 generation; they will not receive direct treatment unless used as a replacement animal.

Identification

Numbering: Unique for each F0 animal and all selected F1 offspring within study. All pre-weaning offspring numbered individually within each litter on Day 1 of age.
Method: Microchip (F0 and selected F1 generation). Toe tattoo (pre-weaning offspring).
Cage labels: Uniquely identifying the occupant(s).

Precommencement Animal Replacement

Eight male and eight female spare F0 animals will be ordered to replace any individuals rejected, following randomization.
Rejection before treatment: Ill-health. Body weight extremes. On Day 1 (before first dose administration) variations in body weight of animals should not exceed ±20% of the mean for each sex.
Replacement during treatment: None scheduled.

Following Study Director approval to remove the spare F0 animals from the study, some of the spare F0 animals will be assigned to the internal sentinel animal program.


Animals - Housing, Diet and Water Supply, and Environmental Enrichment

Environmental Control

Animal facility: Limited access – to minimize entry of external biological and chemical agents.
Air supply: Filtered, not recirculated.
Temperature: Maintained within the range of 20-24ºC.
Relative humidity: Maintained within the range of 40-70%.
Monitored daily. Excursions outside these ranges documented in the study data.

Lighting: 12 hours light : 12 hours dark.
Alarm systems: Activated on ventilation failure and when temperature/humidity limits exceeded.
Electricity supply: Public supply with automatic stand-by generators


Animal Accommodation:
Study period Number of animals/cage Cage material Cage flooring
Male Female
F0 generation (from acclimatization) and selected F1 generation (from weaning) Up to 4 Up to 4 Polycarbonate Solid polycarbonate
Pairing 1 : 1 Polycarbonate Stainless steel grid
Males to termination Up to 4 - Polycarbonate Solid polycarbonate
Females after mating (from Day 0 after mating) - 1 Polycarbonate Solid polycarbonate
Females during littering (from Day 20 after mating) - 1 + litter Polycarbonate Solid polycarbonate
Females to termination (after weaning) - Up to 4 Polycarbonate Solid polycarbonate

Bedding, Diet and Water Supply

Bedding type: Softwood based bark-free fiber, sterilized by autoclaving.
Certification: Certificates of analysis are routinely received from the supplier.

Diet name and type: SDS VRF1 Certified, pelleted diet. A sample (100g) of each batch of diet used will be retained within Pharmacy (frozen, -10 to -30°C). Sample will be discarded after finalization of the report.
Availability: Non-restricted (except overnight for blood sampling for hematology, blood chemistry and thyroid hormone or urinalysis investigations for F0 and F1 Cohort 1A animals).
Certification: Before delivery each batch of diet is analyzed by the supplier for various nutritional components and chemical and microbiological contaminants.
Supplier’s analytical certificates are scrutinized and approved before any batch of diet is released for use.
This diet contains no added antibiotic or other chemotherapeutic or prophylactic agent.

Supply: Potable water from the public supply.
Availability: Non-restricted via polycarbonate bottles with sipper tubes (except overnight for urinalysis investigation).
Certification: Certificates of analysis are routinely received from the supplier.

Environmental enrichment

During the acclimatization and appropriate study periods environmental enrichment in the form of Aspen chew products (soft white untreated wood), Diamond Twists and a plastic shelter will be available in each home cage. Wood-based products will be available during pairing; plastic shelters are not provided during pairing or lactation (from Day 20 after mating)
From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings will be provided to each cage as nesting material.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Treated at: Constant doses in mg/kg/day.
Controls (Group 1): Vehicle at the same volume dose as treated groups.
Volume dose: 4 mL/kg/day
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Frequency: Once daily, at approximately the same time each day.
Sequence: Groups dosed in ascending order.
Formulation: A daily record of the usage of formulation will be maintained based on weights before and after dosing.
Formulations are stirred using a magnetic stirrer before and throughout the dosing procedure.
Administration: Before administration, the required amount of dose formulation is drawn up into the syringe. After the dose has been drawn up the outside of the cannula is wiped clean of formulation residue with a disposable tissue and the end of the cannula will be lightly tapped onto clean tissue to remove any remaining droplets. The cannula will then be dipped into a container filled with 5% glucose solution.
Storage of formulation: Refrigerated (2 to 8°C)
Details on mating procedure:
F0 pairing commences: After 2 weeks of treatment.
F1 Cohort 1B animals: 10 weeks after commencement of formal F1 generation (animals paired at no less than 14 weeks of age)
Male/female ratio: 1:1 (sibling pairing will not be permitted).
Duration of pairing: Up to 2 weeks, no mating change-overs.
Daily checks for evidence of mating: Ejected copulation plugs. Sperm within vaginal smear.
Day 0 of gestation: When positive evidence of mating detected.
Male/female separation: Day when mating evidence detected.
Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
To be reported
Duration of treatment / exposure:
F0 animals For 10 weeks before pairing until termination after litters are weaned. F1 animals From weaning until termination of respective cohort.
Frequency of treatment:
Single dose daily
Details on study schedule:
Litters culled to 10 (where possible 5 males and 5 females) on Day 4 of age.
Formal commencement of the F1 generation is on a nominal Day 28 of age (where possible 28±2 days of age for selected F1 animals).
2 males and 2 females from each litter; Microchipped day 18-21 and separated from littermates on day 21.

F0 adult animals: Females: between Day 22 to Day 25 post partum. Males: After at least 10 weeks of treatment and after weaning of the F1 animals, after confirmation that no further mating is required.
F0/F1 females failing to mate: If an estrus smear is seen following completion of the pairing period animals will be terminated as soon as logistically possible.
If no estrus smear is seen, animals will be terminated on Day 25 after the last day of pairing (where Day 0 is the day of separation from pairing).
F0/F1 females failing to produce a viable litter: On or after Day 25 after positive indication of mating.
F0/F1 females with total litter loss: On the day the last offspring dies
F1 unselected offspring and F2 offspring: On Day 4 and Day 22 of age.
F1 Cohort 1A animals: At approximately 13 weeks of age.
F1 Cohort 1B animals: Males: After weaning of the F2 animals, after confirmation that no further mating is required. Females: between Day 22 to Day 25 post partum.
F1 Cohort 1C: After completion of sexual maturation (approximately 6-8 weeks of age).
F1 Cohort 2A: animals At approximately 11-12 weeks of age.
F1 Cohort 2B: animals At Day 21/22 of age.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
10 mg/kg bw/day
Remarks:
Low Dose
Dose / conc.:
30 mg/kg bw/day
Remarks:
Middle Dose
Dose / conc.:
100 mg/kg bw/day
Remarks:
High dose
No. of animals per sex per dose:
F0: 25
F1 1A: 20
F1 1B: 20
F1 1C: 10
F1 2A: 10
F1 2B: 10
Spares: 2
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Dose Level Selection:

Following earlier reported developmental toxicity in OECD 414 (Envigo study DG70MD) and OECD 422 (Harlan, 0142-0417) studies, a dose range finder (DRF) study (Labcorp Study no 8454920) was performed in preparation for this EOGRTS (OECD 443) to investigate tolerability in new-born offspring and early life stages, as the earlier OECD 422 only evaluated offspring up to LD4. The same high dose level of 125 mg/kg was applied as employed in the previous studies. Two groups of 8 pregnant females were dosed from GD6 until weaning with either 60 or 125 mg/kg bw/d in 4 mL arachis oil/kg bw. The F1 offspring
received direct treatment from weaning until maturation (at approximately week 7 of age following attainment of sexual maturation).
Unexpectedly, 125 mg/kg was not tolerated by the pregnant animals, whereas the earlier full studies (90-day, OECD 422, and OECD 414) showed that 125 mg/kg was sufficiently tolerated with no mortality or significant effect on BW and food consumption. In this DRF, one female was found not pregnant, three females failed to litter (with implantations sites) and one female lost her only offspring on Day 1 post-partum (and had 5 implantations sites). This left only three litters for evaluation at 125 mg/kg/day. Selected F1 animals tolerated 60 or 125 mg/kg/day with minor differences in body weight and food consumption but no effects on clinical condition or macropathology.
Based on the combined information from earlier studies and the results from the DRF, a dose level of 100 mg/kg/day is considered suitable for use as the high dose level in the OECD443 study, with low and intermediate dose levels of 10 and 30 mg/kg/day to provide approximate 3 fold dose intervals.

Blood Chemistry:
Blood sampling of F1 Cohort 1A animals coincides with urine collection and will therefore be eprived of water overnight but will have access to water for a minimum period of one hour prior to blood sampling.

Positive control:
No

Examinations

Parental animals: Observations and examinations:
Animals and their cages:
Visually inspected at least twice daily for evidence of reaction to treatment or ill-health.
Only signs which are indicative of ill-health will be routinely recorded as part of this twice daily health check. Those signs which are not indicative of ill-health will routinely be recorded, as appropriate, as part of the detailed physical examination check or the scheduled pre and post dose checks.

Deviations from normal recorded at the time in respect of:
Nature and severity.
Date and time of onset.
Duration and progress of the observed condition.

Physical examination:
Once each week for all F0 and selected F1 generation animals.
For F0/F1 Cohort 1B females on Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation
A detailed physical examination will be performed at nominally the same time of day on each occasion by an
observer. After removal from the home cage, animals will be assessed for physical condition and behavior during handling. Particular attention will be paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior. Any deviations from normal will be recorded with respect to nature, and, where appropriate, degree of severity.

In addition, detailed observations will be performed on F0 and formal F1 generation animals
to establish and confirm a pattern of signs associated with dosing according to the following
minimum schedule:

Minimum schedule:
• Week 1 - Daily.
• Weeks 2 to 4 - twice weekly (middle and end of the week)
• Week 5 onward - once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 21 of lactation for females).
For selected F1 animals dose observations will be performed daily from Day 21 of age up to the formal start of the F1 generation, thereafter observations will be as detailed above.

Detailed observations will be performed in the treatment period, at the following times during the day:

Dose observation:
• Prior to dosing.
• 1 to 2 hours after completion of dosing.
• As late as possible in the working day.

The above schedule will be amended, as necessary, in the light of signs observed.

Mortality (F0 and F1 Generation)
Premature sacrifice: Animals may be killed for reasons of animal welfare.
Animals found dead, or killed for reasons of animal welfare: A necropsy is performed as soon as possible.

Body Weight (F0 and F1 Generation)
F0 Males:
Day that treatment commences.
Each week.
Before necropsy.

F0 Females:
Day that treatment commences.
Each week until mating detected.
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14 and 21 post partum.
Before necropsy.

F1 Selected Males: Day 21, 23, 25 27* and 29* of age, twice during Week 1 of the F1 generation and weekly thereafter.
Before necropsy.

F1 Selected Females: Day 21, 23, 25 27* and 29* of age, twice during Week 1 of the F1 generation and weekly thereafter.
Cohort 1B after mating:
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14 and 21 post partum.
Before necropsy.


Food Consumption (F0 and F1 generation)
F0 Animals:
Weekly.
Food consumption will not be recorded for males and females during the period when paired for mating but will recommence for males once pairing of all the animals is completed.
For females after mating food consumption schedule will match body weight schedule:
Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18 and 18-20 after mating.
Days 1-4, 4-7, 7-14 and 14-21 of lactation

F1 selected animals:
From nominal Week 4 of age, twice weekly during Week 1 of
the F1 generation and weekly thereafter.
Cohort 1B:
Food consumption will not be recorded for males and females during the period when paired for mating but will recommence for males once pairing of all the animals is completed.
For females after mating food consumption schedule will match body weight schedule:
Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18 and 18-20 after mating.
Days 1-4, 4-7, 7-14 and 14-21 of lactation
Oestrous cyclicity (parental animals):
Dry smears: For 15 days before pairing, using cotton swabs.

Wet smears Daily after pairing until evidence of mating confirmed, using
pipette lavage.
On the day of scheduled necropsy.
For females showing no evidence of mating, following completion of the pairing period the females will be separated from the male and vaginal smearing will continue for up to five days or until the first estrus smear is seen.
• If a female shows an estrus smear during this period, the female will be killed as soon as practically possible and subject to macroscopic examination. If necropsy is not possible on the day of the estrus smear, smears will continue until the morning of necropsy.
• If a female does not show an estrus smear, a wet smear will be taken on the morning of necropsy (Day 25 after removal from pairing where Day 0 = day of removal from pairing)
Sperm parameters (parental animals):
Sperm Analysis - F0 males and F1 Cohort 1A males

Vas deferens (from left side) – each animal in each group:
Sperm sample (where possible at least 200) assessed for motility using a computer assisted sperm analyzer (CASA).
Each animal in each group.
A manual assessment of sperm morphology will be performed. Each animal in Groups 1 and 4#

Cauda epididymis (from left side):
The cauda epididymis will be weighed and homogenized and the number of sperm will be counted using a computer assisted sperm analyzer (CASA). Each animal in Groups 1and 4#.

Testis (from left side):
The testis will be homogenized and the number of homogenization-resistant spermatids will be counted using a computer assisted sperm analyzer (CASA). Each animal in Groups 1 and 4#

Sperm analysis is not routinely performed for animals killed or dying prematurely

# If treatment–related changes are suspected, or at the request of the Sponsor, the
examinations may be extended to all animals of all groups and documented in an amendment
to Study Plan.
Litter observations:
In addition to in-life observations stated in the Parental Animals: Obaservations and Examinations the following will be performed.

Parturition Observations and Gestation Length:
Duration of gestation: Time elapsing between mating and commencement of parturition.
Parturition observations: From Day 20 after mating animals checked three times daily for evidence of parturition. If difficulties observed, noted progress of parturition process monitored. Numbers of live and dead offspring recorded.


Records Made During Littering Phase:
Clinical observations: Observed approximately 24 hours after birth and then daily for evidence of ill-health or reaction to treatment.
On Day 1 of age, all offspring will receive a qualitative assessment of body temperature, state of activity and reaction
to handling.

Litter size: Daily on Days 1-21 of lactation.
Litters culled to 8 (where possible 4 males and 4 females) on Day 4 of age.
All culled offspring macroscopically examined (with thyroid hormone samples collected from 10 litters per group).

Sex ratio: Days 1, 4 (before and after culling) and 21 of age.

Individual offspring body weights:
All offspring: Days 1, 4, 7, 14 and 21 of age.
Unselected F1: Day 22 of age.

Weaning of offspring: Day 21 of age.

Selection of offspring (F1 generation):

Ano-genital distance: Offspring on Day 1 of age.

Nipple count: Male offspring Day 13 of age.


Sexual Maturation (All Selected F1 Generation Animals, excluding Cohort 2B):
Males:
Examined daily from Day 38 of age for the completion of balano-preputial separation.
Body weight recorded on day of completion of separation.

Females:
Examined daily from Day 25 of age until vaginal opening occurs. Body weight recorded on day of vaginal opening.
As Section 8.2.1, for F1 Cohort 1A females only, a wet smear will be taken daily from the day of vaginal opening until first
estrus
Postmortem examinations (parental animals):
Macroscopic Pathology
Complete: All animals, including surplus F1 and F2 offspring culled on Day 4 of age.
Where possible, decedent offspring ≤21 days of age (found dead or welfare kill) will be examined and carcass retained.
F0 and F1 Cohort 1B females:
Implantation site count.
Checks: Retained tissues.

Histology
Processing to slide
Full List: F0 animals and all F1 Cohorts: All animals killed or dying
prematurely.
F0 animals, F1 Cohort 1A, F1 Cohorts 2A and 2B: All
terminal animals of Groups 1 and 4.
Reproductive organs: (testes, epididymides, seminal vesicles, prostate, ovaries, uterus (with cervix and oviducts), vagina) and pituitary. F0: Suspect fertility* animals Groups 2 and 3
Abnormalities only: F0 and F1 Cohort 1A: All terminal animals of Groups 2 and 3.
F1 Cohort 1B: All animals
F1 Cohorts 2A and 2B: All terminal animals of Groups 2 and 3.
Stomach F0 and F1 Cohort 1A: All terminal animals of Groups 2 and 3

Processing to block
Reproductive organs: (testes, epididymides, seminal vesicles, prostate, ovaries, uterus (with cervix and oviducts), vagina) and pituitary. F1 Cohort 1B: All animals
All tissues detailed in Pathology F1 Cohorts 2A and 2B: All terminal animals of Groups 2 and 3

See table for pathology procedures
Postmortem examinations (offspring):
See Postmortem examinations(parental animals)
See Other tables and figures
Statistics:
The relative proportions (%) and cell numbers/spleen of each cell population will be reported.
The proportions of the T, B and NK cells will be expressed as a percentage of lymphocytes,
monocytes and granulocytes as a percentage of the total leukocytes. CD4+
and CD8+ T
lymphocyte subsets will be reported as a percentage of total T lymphocytes. The absolute
cell numbers/spleen will be obtained by performing a back calculation using the percentage
proportions of the cell populations obtained from the cytometer and the total cell count of
each spleen sample.
Percentage data will be reported to two decimal places whilst absolute cell numbers
(cells/spleen) will be reported as whole integers. All statistics [e.g. mean, standard deviation
(SD)] presented in this report will be based on reported numbers from the original database.
The contributing scientist will produce a phase report detailing the methods and results and
this will be included as an Attachment in the main report.
Reproductive indices:
Mating performance
and fertility
Individual data tabulated. Group values calculated for males and females separately for the following:
Percentage mating: (Number animals mating/ Animals paired)* 100

Conception rate: (Number animals achieving pregnancy/ Animals mated)* 100

Fertility index: (Number animals achieving pregnancy/ Animals paired)* 100
Offspring viability indices:
Gestation index Calculated for each group as:
(Number of live litters born/ Number pregnant)* 100

Survival indices (%) Individual litter values calculated for:
Post implantation survival index :
(Total number of offspring born/ Total number uterine implantation sites)* 100

Live birth index :
(Number live offspring on Day 1 after littering/ Total number of offspring born)* 100

Viability index :
(Number live offspring on Day 4 before cull/ Number live offspring on Day 1 after littering)* 100

Lactation index :
(Number live offspring on Day 21 after littering/ Number live offspring on Day 4 (after cull))* 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No signs were observed in association with dose administration and there were no signs at routine physical examination that could be related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were three F0 generation male decedents during the course of this study.

Male No. 32 (Group 2) administered 10 mg/kg/day was euthanized on Day 9 due to poor clinical condition. This animal was observed with clinically limited locomotion. Macroscopically, bilaterally dilatated kidney pelvis was observed and correlated with slight pelvic dilatation seen microscopically. A distended stomach was also observed macroscopically, however without microscopic correlations. On the microscopic examination slight focal fibrosis of the femoral periosteum was observed, this might have contributed to the poor use of the animal’s legs. The major factor contributing to the death of this animal was recorded as “poor clinical condition” due to loss of or limited locomotion.

Male No. 54 (Group 3) administered 30 mg/kg/day was euthanized on Day 43 due to mis-dosing. Clinically, this animal showed general signs of poor clinical condition including irregular and rapid breathing, hunched posture, piloerection, and general thin build conformation. Macroscopically, a perforated esophagus was observed with abnormal content in thoracic cavity and adhesions involving multiple organs; this correlated with microscopic findings of slight to moderate inflammation of the esophagus, lungs (pleura) and heart (epicardium) due to perforation of the esophagus. Microscopic findings were also observed in the stomach (moderate hyperkeratosis and slight epithelial hyperplasia of non-glandular stomach, which were considered treatment related) and thymus (moderate generalized decrease in cellularity). The finding in the stomach correlated grossly with thickening of non-glandular mucosa. The major factor contributing to the death of this animal was recorded as “accidental death” and it was considered to be due to mis-dosing.
Male No. 73 (Group 3) administered 30 mg/kg/day was euthanized for animal welfare reasons on Day 10 due to mis-dosing. This animal swallowed part of the cannula during dosing and showed general signs of poor clinical condition. Macroscopically, abnormal content (cannula) was noted in esophagus and stomach. Microscopically, slight epithelial hyperplasia of non-glandular stomach, considered treatment-related, and minimal cortical apoptosis of thymus were observed. The major factor contributing to the death of this animal was recorded as “accidental death” and it was considered to be due to mis-dosing.
Body weight and weight changes:
not specified
Description (incidence and severity):
Body weight gain at 100 mg/kg/day was high during lactation at approximately 128% of Controls from LD1-21 (p<0.01); body weight gain at 10 or 30 mg/kg/day was unaffected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During Days 1 to 3 of lactation food consumption at 100 mg/kg/day was low at approximately 83% of Controls (p<0.05) and overall food consumption at 100 mg/kg/day was slightly low (-7%) when compared with Controls.

Food consumption at 10 or 30 mg/kg/day was unaffected by treatment.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
not specified
Description (incidence and severity):
Males that received 100 mg/kg/day had high mean red blood cell count (+5%; p<0.01), low mean cell haemoglobin (-3%; p<0.05%), low mean cell volume (-3%; p<0.05), high eosinophil count (+25%; p<0.01) and a high mean platelet count (+13%; p<0.05).

Females that received 100 mg/kg/day had a low hematocrit (-4%; p<0.05), a high red cell distribution width (+9%; p<0.05) and a high basophil count (+67%; p<0.05).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
High potassium levels for males at 30 mg/kg/day (+6%; p<0.05) and males at 100 mg/kg/day (+8%; p<0.01).

Treated females also showed high cholesterol levels: + 25% of Controls at 30 mg/kg/day (p<0.05), +32% of Controls at 30 mg/kg/day (p<0.01) and +54% of Controls at 100 mg/kg/day.

Females that received 30 or 100 mg/g/day had high calcium levels (p<0.05)
Urinalysis findings:
not specified
Description (incidence and severity):
When compared with Controls males that received 100 mg/kg/day had high total urinary protein (+72%; p<0.05) and urinary protein levels (+87%; p<0.01); females at 100 mg/kg/day also had high protein levels (+75%; p<0.05).
Organ weight findings including organ / body weight ratios:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Litter size was unaffected by treatment. Cyclicity was thought to be unaffected by treatment.

The number of litters on Day 1 with a live birth index <100% was high at 100 mg/kg/day when compared with the Controls (6 litters vs 1 litter; p<0.05).

The mean post-implantation index, viability index (PND4) and subsequent lactation indices (PND7,14 and 21) were unaffected by administration of Coco-PFAEO at dose levels up to and including 100 mg/kg/day.
Reproductive function: sperm measures:
not specified
Description (incidence and severity):
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

At 100mg/kg/day, in cauda epididymal sperm count (p<0.01) and total millions (p<0.05) were low hen compared with the concurrent Controls and historical control range. There was also a statistically significant decrease in testicular spermatid count (p<0.01) and total millions (p<0.01) when compared with concurrent Controls with both the Control and Group 4 mean values exceeding the HCD range.

At 100mg/kg/day there was a statistically significant decrease in normal sperm (with a correlating increase in total abnormal sperm) when compared with concurrent Controls (p<0.01) and outside of HCD range. This can be attributed to a statistically significant increase in tail abnormalities (p<0.05), specifically tail bent/kinked (p<0.05).

Sperm motility showed no adverse effects of treatment.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Three females failed to litter; this was considered unrelated to Coco-PFAEO administration during the course of the F0 generation.

Female No. 251 (Group 3) administered 30 mg/kg/day was euthanized on Day 25 of gestation for failure to litter. Macroscopically, thickened non-glandular stomach was observed, which correlated microscopically with slight hyperkeratosis and epithelial hyperplasia of the non-glandular mucosa. These findings were considered treatment related. There were no other significant findings present, this female appeared to be cycling normally and was in diestrus.

Microscopically, implantation sites were not present in the uterus. Male No. 51 (Group 3) administered 30 mg/kg/day paired with this female had minimal focal inflammatory cell infiltrate present in the prostate, however due to minimal severity and focal distribution of this findings it is unlikely that this contributed to failure to litter in this female.
Female No. 255 (Group 3) administered 30 mg/kg/day was euthanized on Day 26 of gestation for failure to litter. This female had no significant macroscopic or microscopic findings, it appeared to be cycling normally and was in proestrus. Microscopically, implantation sites were not present in uterus. Male No. 55 (Group 3) administered 30 mg/kg/day paired with this female had no findings that could be considered to contribute to failure to litter in this female.
Female No. 289 (Group 4) administered 100 mg/kg/day was euthanized on Day 26 of gestation for failure to litter. Macroscopically, thickening of non-glandular stomach was observed and correlated with microscopic finding of minimal hyperkeratosis and epithelial hyperplasia of non-glandular mucosa. These findings were considered treatment related. The vagina was missing for this female on tissue trimming, therefore microscopic examination and cycle staging could not be performed. Microscopically, implantation sites were not present in the uterus. Male No. 89 (Group 4) administered 100 mg/kg/day paired with this female had no findings that could be considered to contribute to failure to litter in this female All other F0 animals survived to their scheduled sacrifice.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
ileum
jejunum
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At routine physical examination animals receiving 30 or 100 mg/kg/day showed an increased incidence of increased salivation and females at 100 mg/kg/day had an increase in the incidence of opaque eye(s).

No signs were observed in association with dose administration.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
On Day 1 of age offspring bodyweight at 100 mg/kg/day was low at approximately 89% of Controls for males and 90% of Controls for females (p<0.05).

At 100 mg/kg/day body weight gain from Day 1 to Day 21 of age was low for both males and females (p<0.01; approximately 85% of Controls for males and 86% of Controls for females) and at weaning (PND21) absolute offspring body weight remained low at approximately 86% of Controls for both males and females (p<0.01).

Offspring body weight and bodyweight change at 10 or 30 mg/kg/day were unaffected by treatment.
Selected F1 males and females at dose levels of 100 mg/kg/day showed low mean body weight and low bodyweight gain at weaning on Day 21 up to Day 25 of age (p<0.01).
At the formal commencement of the F1 generation on nominal Day 28 (+/- 2) of age, the mean bodyweight when compared to controls for selected F1 males treated at 30 mg/kg/day (p<0.05) or 100 mg/kg/day (p<0.01) and for females at 100 mg/kg/day (p<0.01) were low.
Subsequent body weight gain from Day 1 to Day 43 of the F1 generation was low for males receiving 100 mg/kg/day (p<0.01).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males receiving 100 mg/kg/day showed low food consumption over Days 1-36 when compared with Controls; females at 30 or 100 mg/kg/day and males at 30 mg/kg/day showed some minor differences that attained statistical significance, but the overall consumption was similar Controls.
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Dose-dependent response observed for Opacity / Cataract, Nuclear and Opacity / Cataract, Perinuclear for the ocular lens in one or both eyes of the mid and high dose groups.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
There were no differences that were considered of toxicological significance; the statistical significances in the treated groups are related to the low male percent in the Control group rather than an effect of treatment. At 100 mg/kg/day completion of sexual maturation for both males and females were approximately three days later than the concurrent Controls (p<0.01). At 100 mg/kg/day the mean bodyweight for males at completion was low when compared to Controls (p<0.01), whilst mean body weight for treated females was similar to Controls.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Ano-genital distance for male and female offspring on Day 1 of age was unaffected by administration of the test item.
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Organ weights (Cohort 1A) --

When compared with Controls males and females had high body weight relative adrenal weight at 100 mg/kg/day (p<0.01), high heart weight at 100 mg/kg/day (p<0.01), high kidney weight at 30or 100 mg/kg/day (p<0.01), high spleen weigh at all dose levels (p<0.05), high thyroid and parathyroid weight at 100 mg/kg/day (p<0.01).

In addition, females at 30 or 100 mg/kg/day had high absolute and body weight relative mean ovarian weights (p<0.01).

Other statistical significances for male animals were attributed to the effect on terminal body weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A -- Macroscopic examinations revealed a high incidence of opaque eyes for both males and females that received 100 mg/kg/day.

There was a dose-related incidence of thickened stomach for both males and females that received 30 or 100 mg/kg/day with one female at 10 mg/kg/day also showing this abnormality.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
ophthalmological examination
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology

Target system / organ toxicity (F1)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
System:
eye
Organ:
lens
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
ileum
jejunum
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables
























 



Control



Coco-PFAEO



Dose Group



1



2



3



4



Dose (mg/kg/day)



0



10



30



100



 












































































































































































































































































































































































































































































 



 



Number of animals affected



Structure



Observation



Group/Sex:



1M



2M



3M



4M



 



1F



2F



3F



4F



 



 



No. Examined:



20



20



20



20



 



20



20



20



20



 



 



 



 



 



 



 



 



 



 



 



 



 



Globe / Orbit



Megalogobus



0



0



0



1



 



0



0



0



0



 



 



 



 



 



 



 



 



 



 



 



 



 



Cornea



Opacities, Superficial



2



5



3



0



 



1



2



2



0



 



 



 



 



 



 



 



 



 



 



 



 



 



Iris



Iritis / Uveitis



0



0



0



0



 



0



0



0



1



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Irregular Pupil



0



0



0



1



 



0



0



0



0



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Miosis



0



0



0



1



 



0



0



0



1



 



 



 



 



 



 



 



 



 



 



 



 



 



Lens



Hyaloid Remnant



0



1



0



0



 



2



0



0



0



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Opacity / Cataract, Anterior suture line



0



0



0



5



 



0



0



0



0



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Opacity / Cataract, Nuclear



0



0



1



13



 



0



0



4



14



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Opacity / Cataract, Perinuclear



0



0



0



2



 



0



0



2



6



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Opacity / Cataract, Posterior cortical



0



0



0



0



 



0



0



0



3



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Opacity / Cataract, Posterior subcapsular



0



0



0



0



 



0



0



1



0



 



 



 



 



 



 



 



 



 



 



 



 



 



Lens



Persistent Hyaloid Artery



0



0



0



0



 



1



0



0



0



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Prominent Nucleus



1



0



2



0



 



0



0



2



0



 



 



 



 



 



 



 



 



 



 



 



 



 



Vitreous



Opacity



0



0



0



1



 



0



0



0



0



 



 



 



 



 



 



 



 



 



 



 



 



 



Optic Nerve



Optic Disc Coloboma



1



0



0



0



 



0



0



0



0



 


Applicant's summary and conclusion

Conclusions:
The current information represents an interim report of OECD 443 EOGRTS on 2, 2’-(C12-18 evennumbered alkyl imino) diethanol, CAS No 71786-60-2. These results are not complete and have not been finalized and should be examined as such when drawing conclusions.

While no finalized conclusions can be yet obtained from the data there are indication of general toxicity seen on the local site of contact in the gastrointestinal tract. Possible evidence of reproductive toxicity is present in F0 males showing decreased sperm count and increased incidence of abnormal sperm in 100 mg/kg bw/day. Cohort 1A also showed possible implications of developmental toxicity in the ophthalmological investigations revealing a dose-dependent response in nuclear and peri-nuclear cataracts down to LOEL of 30 mg/kg bw/day.
Executive summary:


This report describes anatomic pathology findings for animals during and at the end of the dosing phase for Labcorp Study 8453873. The purpose of this study was to assess the influence of Ethanol, 2,2’-iminibos-, N-C12-18-alkyl derives (hereafter referred to as Coco-PFAEO) on reproductive performance when administered by oral gavage to Han Wistar rats. Animals were administered 10, 30 and 100 mg/kg/day Coco-PFAEO.


2-Week Premating (F0)


Clinical Signs


There were no signs at routine physical examination that could be related to administration of Coco-PFAEO.


Dose signs
No signs were observed in association with dose administration


Body Weight
During Week 1 of treatment males receiving 100 mg/kg/day showed significantly low body weight gain at 22% of Controls (p<0.01), subsequent weight gain during Week 2 was similar to Controls.


Conversely females at 100 mg/kg/day showed high bodyweight gain at approximately 180% of Controls (p<0.05) with subsequent weight gain similar to Controls.


Body weight gain at 10 or 30 mg/kg/day was unaffected by treatment.


Food Consumption


When compared with Controls males receiving 100 mg/kg/day had low food consumption during both Week 1 (p<0.01) and Week 2 (p<0.05) of treatment. Females at 100 mg/kg/day has slightly low food consumption during Week 1 at approximately 94% of Controls (p<0.05) and mean consumption during Week 2 was similar to Controls.


Food consumption at 10 or 30 mg/kg/day was unaffected by treatment.


Gestation (F0)


There were no signs at routine physical examination that could be related to treatment.  Increased salivation was observed in association with dose administration for one female at 100 mg/kg/day


Body weight gain during gestation was unaffected by administration of the test item.


Food consumption during gestation was unaffected by administration of the test item.


The following females failed to litter :


• Group 3 no. 251
• Group 3 no. 255
• Group 4 no. 289


Uterine examination of these females at necropsy did not show any implantation sites.


Litter size was unaffected by treatment.


Lactation (F0 - F1)


No signs were observed in association with dose administration and there were no signs at routine physical examination that could be related to treatment.


During Days 1 to 3 of lactation food consumption at 100 mg/kg/day was low at approximately 83% of Controls (p<0.05) and overall food consumption at 100 mg/kg/day was slightly low (-7%) when compared with Controls.


Food consumption at 10 or 30 mg/kg/day was unaffected by treatment.


The number of litters on Day 1 with a live birth index <100% was high at 100 mg/kg/day when compared with the Controls (6 litters vs 1 litter; p<0.05).


The mean post-implantation index, viability index (PND4) and subsequent lactation indices (PND7,14 and 21) were unaffected by administration of Coco-PFAEO at dose levels up to and including 100 mg/kg/day.


There were no differences that were considered of toxicological significance; the statistical significances in the treated groups are related to the low male percent in the Control group rather than an effect of treatment.


On Day 1 of age offspring bodyweight at 100 mg/kg/day was low at approximately 89% of Controls for males and 90% of Controls for females (p<0.05).


At 100 mg/kg/day body weight gain from Day 1 to Day 21 of age was low for both males and females (p<0.01; approximately 85% of Controls for males and 86% of Controls for females) and at weaning (PND21) absolute offspring body weight remained low at approximately 86% of Controls for both males and females (p<0.01).


Offspring body weight and bodyweight change at 10 or 30 mg/kg/day were unaffected by treatment.


Fertility (F0)


Three females failed to litter; this was considered unrelated to Coco-PFAEO administration during the course of the F0 generation.


Female No. 251 (Group 3) administered 30 mg/kg/day was euthanized on Day 25 of gestation for failure to litter. Macroscopically, thickened non-glandular stomach was observed, which correlated microscopically with slight hyperkeratosis and epithelial hyperplasia of the non-glandular mucosa. These findings were considered treatment related. There were no other significant findings present, this female appeared to be cycling normally and was in diestrus. Microscopically, implantation sites were not present in the uterus. Male No. 51 (Group 3) administered 30 mg/kg/day paired with this female had minimal focal inflammatory cell infiltrate present in the prostate, however due to minimal severity and focal distribution of this findings it is unlikely that this contributed to failure to litter in this female.
Female No. 255 (Group 3) administered 30 mg/kg/day was euthanized on Day 26 of gestation for failure to litter. This female had no significant macroscopic or microscopic findings, it appeared to be cycling normally and was in proestrus. Microscopically, implantation sites were not present in uterus. Male No. 55 (Group 3) administered 30 mg/kg/day paired with this female had no findings that could be considered to contribute to failure to litter in this female.
Female No. 289 (Group 4) administered 100 mg/kg/day was euthanized on Day 26 of gestation for failure to litter. Macroscopically, thickening of non-glandular stomach was observed and correlated with microscopic finding of minimal hyperkeratosis and epithelial hyperplasia of non-glandular mucosa. These findings were considered treatment related. The vagina was missing for this female on tissue trimming, therefore microscopic examination and cycle staging could not be performed. Microscopically, implantation sites were not present in the uterus. Male No. 89 (Group 4) administered 100 mg/kg/day paired with this female had no findings that could be considered to contribute to failure to litter in this female
All other F0 animals survived to their scheduled sacrifice.


 


Terminal summary (F0)


No test item-related mortality occurred during the course of the F0 generation. There were three male decedents euthanized for welfare reasons due to mis-dosing and poor clinical condition. Three females failed to litter; this was considered unrelated to Coco-PFAEO administration.
At the terminal sacrifice of F0 generation, Coco-PFAEO-related absolute and body weight relative mean organ weight increases were noted for the liver, kidneys and adrenal glands, without macroscopic or microscopic correlations.
At the terminal sacrifice of F0 generation, Coco-PFAEO-related microscopic findings were observed in the non-glandular stomach. Hyperkeratosis and epithelial hyperplasia of non-glandular stomach were observed in males and females administered 30 or 100 mg/kg/day, and in males administered 10 mg/kg/day, with slightly higher incidence present in males. This correlated with thickened non-glandular stomach observed macroscopically across all dose levels in males and in females administered 30 or 100 mg/kg/day. Hyperkeratosis often occurs in association with hyperplasia of underlying epithelium, they are considered to be adaptive responses most likely related to the test item’s irritating effect. There were no associated inflammatory and necrotic changes present. Due to higher severity and incidence these changes were considered adverse in males and females administered 100 mg/kg/day, and non-adverse in animals administered 10 or 30 mg/kg/day as they occurred at lower incidence and severity, when compared with high dose animals.


There were three F0 generation male decedents during the course of this study.


Male No. 32 (Group 2) administered 10 mg/kg/day was euthanized on Day 9 due to poor clinical condition. This animal was observed with clinically limited locomotion. Macroscopically, bilaterally dilatated kidney pelvis was observed and correlated with slight pelvic dilatation seen microscopically. A distended stomach was also observed macroscopically, however without microscopic correlations. On the microscopic examination slight focal fibrosis of the femoral periosteum was observed, this might have contributed to the poor use of the animal’s legs. The major factor contributing to the death of this animal was recorded as “poor clinical condition” due to loss of or limited locomotion.


Male No. 54 (Group 3) administered 30 mg/kg/day was euthanized on Day 43 due to mis-dosing. Clinically, this animal showed general signs of poor clinical condition including irregular and rapid breathing, hunched posture, piloerection, and general thin build conformation. Macroscopically, a perforated esophagus was observed with abnormal content in thoracic cavity and adhesions involving multiple organs; this correlated with microscopic findings of slight to moderate inflammation of the esophagus, lungs (pleura) and heart (epicardium) due to perforation of the esophagus. Microscopic findings were also observed in the stomach (moderate hyperkeratosis and slight epithelial hyperplasia of non-glandular stomach, which were considered treatment related) and thymus (moderate generalized decrease in cellularity). The finding in the stomach correlated grossly with thickening of non-glandular mucosa. The major factor contributing to the death of this animal was recorded as “accidental death” and it was considered to be due to mis-dosing.


Male No. 73 (Group 3) administered 30 mg/kg/day was euthanized for animal welfare reasons on Day 10 due to mis-dosing. This animal swallowed part of the cannula during dosing and showed general signs of poor clinical condition. Macroscopically, abnormal content (cannula) was noted in esophagus and stomach. Microscopically, slight epithelial hyperplasia of non-glandular stomach, considered treatment-related, and minimal cortical apoptosis of thymus were observed. The major factor contributing to the death of this animal was recorded as “accidental death” and it was considered to be due to mis-dosing.


At the terminal sacrifice, statistically significant Coco-PFAEO-related organ weight increases were noted for the kidneys, liver and adrenal glands.


The absolute and body weight relative mean kidney weights of F0 generation males administered 30 or 100 mg/kg/day were statistically significantly higher than in controls, however without obvious dose response and macroscopic or microscopic correlations.
The body weight relative mean liver weights of F0 generation males and females administered 100 mg/kg/day were statistically significantly higher than in controls, with absolute liver weights being higher only in females administered 100 mg/kg/day, without macroscopic or microscopic correlations.


The absolute and body weight relative mean adrenal weights of F0 generation females administered 100 mg/kg/day were statistically significantly higher than in controls, without macroscopic or microscopic correlations.


All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.


At the terminal sacrifice, macroscopic findings related to treatment with Coco-PFAEO were observed in the non-glandular stomach.


Thickening of the non-glandular stomach was observed in males at all dose levels and in females administered 30 or 100 mg/kg/day, this correlated microscopically with hyperkeratosis and epithelial hyperplasia of non-glandular stomach.


Dark areas of the glandular mucosa of the stomach were also observed in a few females across the treated groups, this correlated microscopically with mucosal congestion/hemorrhage; this was also observed in a single male administered 10 mg/kg/day, however without microscopic correlations. Due to minimal severity, low incidence, and occurrence only in one sex, this finding was considered to be incidental. All other macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for rats of this age and/or strain. Therefore, they were considered not test item related.


At the terminal sacrifice Coco-PFAEO-related microscopic findings were observed in the non-glandular stomach.


In the non-glandular stomach, mostly diffuse minimal to marked hyperkeratosis and minimal to moderate epithelial hyperplasia of the non-glandular mucosa were observed in males and females administered 30 or 100 mg/kg/day, and in males administered 10 mg/kg/day, with the slightly higher incidence present in males. This correlated with thickened non-glandular stomach observed macroscopically across all dose levels in males and in females administered 30 or 100 mg/kg/day and was most likely related to the test item’s irritant effect.


The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
In the kidneys, renal tubular carcinoma was observed in one control female (No. 208). This neoplasm may occur spontaneously, particularly in old animals. All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for rats at this age; therefore, they were considered not test item related.


At 100mg/kg/day, in cauda epididymal sperm count (p<0.01) and total millions (p<0.05) were low hen compared with the concurrent Controls and historical control range. There was also a statistically significant decrease in testicular spermatid count (p<0.01) and total millions (p<0.01) when compared with concurrent Controls with both the Control and Group 4 mean values exceeding the HCD range.


At 100mg/kg/day there was a statistically significant decrease in normal sperm (with a correlating increase in total abnormal sperm) when compared with concurrent Controls (p<0.01) and outside of HCD range. This can be attributed to a statistically significant increase in tail abnormalities (p<0.05), specifically tail bent/kinked (p<0.05).


Sperm motility showed no adverse effects of treatment.


Maturation (F1)


At routine physical examination animals receiving 30 or 100 mg/kg/day showed an increased incidence of increased salivation and females at 100 mg/kg/day had an increase in the incidence of opaque eye(s).


No signs were observed in association with dose administration.


Selected F1 males and females at dose levels of 100 mg/kg/day showed low mean body weight and low bodyweight gain at weaning on Day 21 up to Day 25 of age (p<0.01).
At the formal commencement of the F1 generation on nominal Day 28 (+/- 2) of age, the mean bodyweight when compared to controls for selected F1 males treated at 30 mg/kg/day (p<0.05) or 100 mg/kg/day (p<0.01) and for females at 100 mg/kg/day (p<0.01) were low.
Subsequent body weight gain from Day 1 to Day 43 of the F1 generation was low for males receiving 100 mg/kg/day (p<0.01).


Males receiving 100 mg/kg/day showed low food consumption over Days 1-36 when compared with Controls; females at 30 or 100 mg/kg/day and males at 30 mg/kg/day showed some minor differences that attained statistical significance, but the overall consumption was similar Controls.


Ano-genital distance for male and female offspring on Day 1 of age was unaffected by administration of the test item.


At 100 mg/kg/day completion of sexual maturation for both males and females were approximately three days later than the concurrent Controls (p<0.01). At 100 mg/kg/day the mean bodyweight for males at completion was low when compared to Controls (p<0.01), whilst mean body weight for treated females was similar to Controls.


Terminal Summary (F1)


Ophthalmic examination revealed lens opacity for males and females receiving 30 or 100 mg/kg/day compared with Controls; a dose response was apparent.


When compared with Controls males and females had high body weight relative adrenal weight at 100 mg/kg/day (p<0.01), high heart weight at 100 mg/kg/day (p<0.01), high kidney weight at 30or 100 mg/kg/day (p<0.01), high spleen weigh at all dose levels (p<0.05), high thyroid and parathyroid weight at 100 mg/kg/day (p<0.01).


In addition, females at 30 or 100 mg/kg/day had high absolute and body weight relative mean ovarian weights (p<0.01).


Other statistical significances for male animals were attributed to the effect on terminal body weight.


Macroscopic examinations revealed a high incidence of opaque eyes for both males and females that received 100 mg/kg/day.


There was a dose-related incidence of thickened stomach for both males and females that received 30 or 100 mg/kg/day with one female at 10 mg/kg/day also showing this abnormality.