2,2'-(C16-18 (evennumbered) alkyl imino) diethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(C16-18 (evennumbered) alkyl imino) diethanol

EC Number:

620-539-0

Cas Number:

1218787-30-4

Molecular formula:

No molecular formula

IUPAC Name:

2,2'-(C16-18 (evennumbered) alkyl imino) diethanol

Details on test material:

- Chemical name: 2,2'-(C16-18 (evennumbered) alkyl imino) diethanol
- EC number: 620-539-0

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:

Han Wistar (RccHan®:WIST) rat
Supplier: Envigo RMS (UK) Ltd.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number of animals ordered 44 males and 48 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Males: Six days before commencement of treatment.
Females: 20 days before commencement of treatment.
Age of the animals at the start of treatment Males 84 to 90 days old.
Females 98 to 104 days old.
Weight range of the animals at the start of treatment Males 315 to 379 g. Females 208 to 245 g.

3.3.2 Allocation and Identification
Allocation On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.
On Day 1 of study all animals were weighed, and body weights were reviewed by Study Management. Body weight of animals did not exceed 20% of the mean for each sex.
Identification of animals Each adult animal was assigned a number and identified uniquely within the study by a microchip before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

3.4.1 Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24C and 40-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

3.4.2 Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless-steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used throughout the study except during pairing.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding Solid bottom cages contained softwood-based, bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage
Pre-pairing up to four animals of one sex
Pairing one male and one female
Males after mating up to four animals
Gestation one female
Lactation one female + litter

3.4.3 Environmental Enrichment
Aspen wood-based products A soft white untreated wood block was provided to each cage throughout the study (except during lactation) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.
Paper shavings Approximately two handfuls of paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation. Shavings were replaced at the same frequency as the bedding.
Diamond twists Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

3.4.4 Diet Supply
Diet SDS VRF1 Certified pelleted.
A sample (100 g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30C) until finalization of the report. Samples will be discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

3.4.5 Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Route: Oral gavage using a suitably graduated syringe and a flexible cannula inserted via the mouth.

Vehicle:

arachis oil

Details on oral exposure:

Treated at: Constant doses in mg/kg/day.

Volume dose: 4 mL/kg body weight for Groups 1-3 and Group 4 female (and Group 4 male until Day 11).
3 mL/kg body weight for Group 4 males from Day 12 onwards.

Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the volume dose of 4 mL/kg body weight.

Frequency: Once daily at approximately the same time each day.

Method: Before administering the test substance, the required amount of dose formulation was drawn up into the syringe. After the dose had been drawn up the outside of the catheter was wiped clean of formulation residue with a disposable tissue and the end of the catheter was lightly tapped onto clean tissue to remove any remaining droplets. The catheter was then dipped into a container filled with 5% glucose solution.

Formulation: A daily record of the usage of formulation was maintained based on weights.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity The homogeneity and stability of formulations during storage were determined as part of another study, Labcorp GLP Study No. 8470219. In that study formulations in the range 1 to 100 mg/mL were determined to be stable for:
• One day at ambient temperature (15 to 25C)
• 15 days when stored refrigerated (2 to 8C)
Achieved concentration Samples of each formulation prepared for administration in the first and last week of treatment were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

From 2 weeks premating till scheduled terminal sacrifice on day 13 of lactation for Females (F0)
From 2 weeks premating till scheduled terminal sacrifice in week 5 , at least 4 weeks, for Males (F0)

Frequency of treatment:

Once daily by oral gavage for all F0 animals.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

F0 Males only

Doses / concentrations 5

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

F0 Females and Offspring only

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels for this study were selected based on the results of a two-phase preliminary study conducted at these laboratories (Labcorp Study No. 8470220), where Han Wistar rats received Ethomeen HT/12 at doses of 150, 175 or 200 mg/kg/day, for 14 days in an initial toxicity phase followed by an embryo-fetal phase lasting 14 days, consisting of time-mated females dosed at 125 or 175 mg/kg/day.
In males, doses of 150, 175 or 200 mg/kg/day were not tolerated, resulting in body weight loss and reduced food intake with no dose-response, and a premature death of two males receiving 175 mg/kg/day and two males receiving 150 mg/kg/day. Females were affected to a lesser extent than males. At the macroscopic examination, the targets of toxicity were determined to be the gastro-intestinal tract (principally manifest as thickening of the small intestine and stomach and, in males only, stomach depressions) and mesenteric lymph nodes (pallor) in both sexes, which was consistent with other compounds of this type.
Although the maximum tolerated dose could not be determined in Study No. 8470220, a high dose of 100 mg/kg/day was selected as the high dose for this study. Experience with similar compounds suggests that this was likely to be tolerated, however in the event that marked body weight loss and/or adverse clinical signs or mortality occur, then the highest dose may be lowered to 75 mg/kg/day by an amendment to the study plan. The low and intermediate dose levels are set at 10 and 30 mg/kg/day, respectively, with an approximate three-fold ratio between doses.
Body weight losses occurred in males receiving 100 mg/kg/day, with one male being prematurely sacrificed due to the extent of the body weight loss. Therefore, from Day 12 of dosing, the dose level was reduced to 75 mg/kg/day for Group 4 males.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 13 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer who was unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sacrifice and pathology:

3.6 Terminal Investigations

3.6.1 Method of Kill:
All adult animals Carbon dioxide asphyxiation with subsequent exsanguination (there was no exposure to carbon dioxide occurred until after the completion of blood sampling for thyroid hormone assays).
Offspring - selected for thyroid hormone sampling on Day 4 and 13 of age Decapitation.
Offspring - not selected for thyroid hormone sampling Intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.


3.6.2 Necropsy:
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Time of Necropsy:
F0 males During Week 5 - after at least four weeks of treatment.
F0 females failing to produce a viable litter Day 25 after mating.
F0 females whose litter died before Day 13 On or after day the last offspring died.
F0 females Day 13 of lactation.
F1 offspring Selected offspring for thyroid hormone analysis - Day 4 of age
Scheduled kill - Day 13 of age.

Pathology procedures for the five lowest numbered surviving F0 males and first five surviving lactating F0 females per group at scheduled termination, plus all F0 decedent animals

All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormalities retained in appropriate fixative.

Thyroid glands were preserved from one male and one female in each litter, where possible.


3.6.3 Organ Weights
For bilateral organs, left and right organs were weighed together. The required organs were weighed for animals killed at scheduled termination.


3.6.4 Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes Initially in modified Davidson’s fluid.
Eyes In Davidson’s fluid.


3.6.5 Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List The five lowest F0 males and the first five lactating females with a surviving litter in Groups 1 and 4 at scheduled termination.
All animals killed or dying prematurely.
Mesenteric lymph nodes(s), jejunum and ileum The five lowest numbered surviving males and the first five lactating females with a surviving litter in Groups 2 and 3 at scheduled termination.
Abnormalities only All animals.

Routine staining Sections were stained with hematoxylin and eosin; in addition, samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.


3.6.6 Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Premature deaths All F0 animals in Groups 1 and 4. All specified in Section 3.6.
Scheduled kill Five lowest numbered F0 males and first five surviving lactating females in Groups 1 and 4. All specified in Section 3.6.
All F0 animals. Abnormalities only.
Five lowest numbered F0 males and first five surviving lactating F0 females in Groups 2 and 3. Mesenteric lymph node(s), jejunum and ileum

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Statistics:

Yes

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The appearance and behavior of the animals were unaffected by treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were five unscheduled deaths in this study, one of which was considered to be treatment-related.
Group 4 male (No. 31), given 100 mg/kg/day, was euthanized because of acute weight loss on Day 11. The macroscopic examination revealed abnormally pale contents of the ileum, cecum, colon and rectum; thickening of the duodenum and jejunum which correlated with infiltration of foamy macrophages in the intestinal mucosa, and abnormally pale coloration of stomach, which correlated with hyperplasia of the nonglandular region. There was also abnormal pale coloration of the jejunum and mesenteric lymph nodes which correlated with the macrophage infiltrates in the jejunal mucosa and intrasinusoidal region of the mesenteric lymph node, respectively. The major factor of death was considered to be poor clinical condition and this death was considered treatment-related.
Group 4 female (No. 138), given 100 mg/kg/day, was found dead on Gestation Day 1. The macroscopic examination included thickening of the esophagus, which correlated microscopically with moderate multifocal pyogranulomatous inflammation of the esophageal serosa with multiple bacterial colonies, fibrous pale adhesions involving multiple organs and clear abnormal fluid in the thoracic cavity which correlated microscopically with moderate multifocal pyogranulomatous inflammation of the serosa with presence of food material. There was also slight mixed inflammation of the epicardium of the heart, which was related to the adhesions. These findings were attributed to gavage misdosing. The small spleen correlated with slight decreased cellularity of the spleen and the enlarged adrenal glands correlated with slight diffuse cortical hypertrophy of the adrenals, and these findings were attributed to stress. The major cause of death was considered to be accidental death due to misdosing and this death was considered not treatment-related.
Group 3 female (No. 110), given 30 mg/kg/day, was euthanized on Day 25 due to failure to litter. There were no macroscopic or microscopic findings.
Group 4 female (No. 134), given 100 mg/kg/day was euthanized on Day 3 due to total litter loss. The macroscopic examination included multifocal pale areas of the lungs correlating with microscopic slight multifocal alveolar aggregation and pale inactive mammary gland which correlated with microscopic slight secretory activity. These findings were incidental and considered not treatment-related.
Group 4 female (No. 139), given 100 mg/kg/day, was euthanized on Day 25 due to failure to litter. There were no macroscopic findings. This female was not examined microscopically.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, there was an initial loss of body weight at all doses, with the extent of the loss being greatest at 100 mg/kg/day. Thereafter (Day 4-11), body weight loss persisted in males receiving 100 mg/kg/day, although to a lesser extent than during Day 1-4, and weight gain was similar to control in males receiving 10 or 30 mg/kg/day. The high dose level was reduced to 75 mg/kg/day from Day 12 onwards; from Day 11-36, mean body weight gain was only 11% lower than control. Consequently, overall body weight gain (Day 1-36) was reduced by 20, 11 and 70%, compared with control, in males receiving 10, 30 or 100/75 mg/kg/day, respectively.
In females before pairing, an initial loss of body weight occurred from Day 1-4 at 100 mg/kg/day and gain was slightly lower than control at 30 mg/kg/day. Thereafter (Day 4-15), body weight gain was slightly higher than control at 10 or 30 mg/kg/day but was 16% lower than control at 100 mg/kg/day. Overall body weight gain before pairing (Day 1-15) was unaffected by treatment at 10 or 30 mg/kg/day but was 46% lower than control at 100 mg/kg/day.
During gestation, body weight gain was unaffected from Gestation Day 0-11, but was lower than control from Gestation Day 11-20 in females receiving 100 mg/kg/day (19% reduction), resulting in overall body weight gain during gestation (Gestation Day 0-20) that was 13% lower than control at 100 mg/kg/day.
During lactation, body weight gain was persistently higher than control in females receiving 100 mg/kg/day, with statistical significance being attained for the overall difference (38% increase).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a reduction of food intake from (Day 1-15), compared with control, in males receiving 100/75 mg/kg/day (33% lower than control) but thereafter, following the lowering of the high dose level from Day 12, food intake was similar to control at all dose levels from Day 22-36. In females before pairing, overall food intake (Day 1-15) was 15% lower than control at 100 mg/kg/day. Overall food intake during gestation (Gestation Day 0-20) was marginally, but statistically significantly, lower than control in females receiving 100 mg/kg/day (12% reduction, which in terms of the amount consumed equated to only 2 g less).
Food intake was unaffected in either sex receiving 10 or 30 mg/kg/day or during lactation in females receiving 100 mg/kg/day.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

All values were within historical control ranges and thus attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma performed at termination revealed, compared with controls, high bile acid concentration in males receiving 100/75 mg/kg/day and females receiving 100 mg/kg/day.

Endocrine findings:

no effects observed

Description (incidence and severity):

The majority of individual values were within the historical control range (98-percentile range 31992-59420 pg/mL; n=69) except for two males receiving 100/75 mg/kg/day (Nos. 35 (68000 pg/mL) and 36 (63400 pg/mL)).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

At the motor activity assessment for males in Week 5, there was a treatment-related reduction of total high (rearing) beam breaks in males receiving 30 or 100/75 mg/kg/day. Statistical significance was not obtained.
There was no indication of neurotoxicity in the weekly arena observations, sensory reactivity or grip strength. This finding was attributed to effects in the GI tract and lower body weights.

All values were within historical control ranges and thus attributed to normal biological variation.

Immunological findings:

no effects observed

Description (incidence and severity):

All values were within historical control ranges and thus attributed to normal biological variation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test article-related, lower than control organ weights were noted for the prostate for males at 100/75 mg/kg/day. These weights were atrributed to stress and lower bodyweight gain and not directly from the substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related macroscopic findings included thickening of the jejunum in one male (No.37) at 100/75 mg/kg/day.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Sensory reactivity and grip strength were unaffected by treatment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings included decreased general cellularity and increased cellularity of intrasinusoidal macrophages of the mesenteric lymph nodes, foamy macrophage infiltrates within the mucosa of the jejunum and ileum of males at 100/75 mg/kg/day or females at 100 mg/kg/day. Thickening of the jejunum in one male (No.37) at 100/75 mg/kg/day correlated with increased foamy macrophage infiltrate in the mucosa.
Increased cellularity of intrasinusoidal macrophages of the mesenteric lymph nodes and foamy macrophage infiltrates within the mucosa of the jejunum were also seen in males and females at 30 mg/kg/day. The foamy macrophage infiltrates of mesenteric lymph node, the jejunal and ileal mucosa were characterized microscopically by increased numbers and size of macrophages with a vacuolated cytoplasm. The presence of foamy macrophages was considered to represent increased phagocytic activity of the test article. Decreased cellularity of the mesenteric lymph nodes was attributed to the test article due to increased mobilization of inflammatory cells into the intestinal mucosa. Dilated/cystic sinuses were also seen in mesenteric lymph nodes of males at 100/75 mg/kg/day.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

> 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Highest dose tested

2

Key result

Dose descriptor:

NOAEL

Effect level:

> 30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Target system / organ toxicity

1

Critical effects observed:

no

Lowest effective dose / conc.:

30 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

ileum

jejunum

mesenteric lymph node

2

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (actual dose received)

System:

other: Body wieght

Organ:

other: Body weight

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

It is concluded that the oral administration of Ethomeen HT/12 to Han Wistar rats at initial dose levels of 10, 30 or 100 mg/kg/day was tolerated in females, but due to acute weight loss and premature death, 100 mg/kg/day was not tolerated by males and the dose was reduced to 75 mg/kg/day from Day 12 onward for the remainder of the study period.

There were microscopic pathology findings in the mesenteric lymph nodes and gastro-intestinal tract of males receiving 100/75 mg/kg/day, females receiving 100 mg/kg/day and both sexes receiving 30 mg/kg/day which were considered non-adverse (decreased general cellularity and increased cellularity of intrasinusoidal macrophages of the mesenteric lymph nodes, foamy macrophage infiltrates within the mucosa of the jejunum and ileum). Lower weight gain/body weight loss occurred in males receiving 100/75 mg/kg/day and was considered adverse.

Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment, and despite a statistically significance increase of serum thyroxine concentration in males receiving 30 or 100/75 mg/kg/day, there were no effects on thyroid gland weights and there were no microscopic findings in the thyroid gland. In the context of this study, Ethomeen HT/12 showed no evidence of being an endocrine disruptor.

Due to the adverse effect on body weight gain in males receiving 100/75 mg/kg/day, the no-observed-adverse-effect level (NOAEL) of Ethomeen HT/12 for systemic toxicity was considered to be 30 mg/kg/day for males and at least 100 mg/kg/day for females. The NOAEL for reproductive/developmental toxicity was at least 100 mg/kg/day for F0 females and offspring, and at least 75 mg/kg/day for reproductive toxicity for F0 males.

Executive summary:

The purpose of this study was to assess the general systemic toxic potential of Ethomeen HT/12 when administered orally, by gavage, to Han Wistar rats for at least four weeks, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints. Dose levels of 10, 30 or 100 mg/kg/day were assessed, but due to significant body weight losses occurring in males receiving 100 mg/kg/day, with one male being prematurely sacrificed due to the extent of the body weight loss, the highest dose for males was lowered to 75 mg/kg/day from Day 12 of dosing. The highest dose remained 100 mg/kg/day for females.

Parental males and females receiving 100/75 of 100 mg/kg/day, respectively, showed persistently lower mean body weights than controls for the majority of the study period. Although the overall weight gain was lower than control in males receiving 10 or 30 mg/kg/day, this was largely attributed to initial weight loss, as weight gain was similar to control from Day 4 onwards and this was therefore considered non-adverse. Additionally, reduced food intake was also observed in males receiving 100/75 mg/kg/day though this was not consistent over the full study period. Reduced body weight gain was considered adverse in males receiving 100/75 mg/kg/day.

 

The target of toxicity was considered localized to the gastro-intestinal tract. Treatment-related microscopic findings included decreased general cellularity and increased cellularity of intrasinusoidal macrophages of the mesenteric lymph nodes, foamy macrophage infiltrates within the mucosa of the jejunum and ileum of males at 100/75 mg/kg/day or females at 100 mg/kg/day. Increased cellularity of intrasinusoidal macrophages of the mesenteric lymph nodes and foamy macrophage infiltrates within the mucosa of the jejunum were also seen in males and females at 30 mg/kg/day. The presence of foamy macrophages was considered to represent increased phagocytic activity, related to the test article. Dilated/cystic sinuses were also seen in mesenteric lymph nodes of males at 100/75 mg/kg/day. These findings were the likely cause of the alterations to leucocyte counts identified at the clinical pathology investigations. All microscopic findings were considered non-adverse but the changes in the jejunum and ileum may, in some instances, be correlated to the reduced food intake and weight gain/body weight loss reported.

 

Although the mean absolute and relative weight of the prostate gland was lower than control in males receiving 100/75 mg/kg/day, no treatment-related histopathological findings were identified in the reproductive organs of any animal. The lower prostate weight was attributed to the stress resulting from reduced weight gain and food intake and was not directly related to treatment.

 

At the motor activity assessment for males in Week 5, there was a treatment-related reduction of total high (rearing) beam breaks in males receiving 30 or 100/75 mg/kg/day and for total low (cage-floor) beam breaks in males receiving 100/75 mg/kg/day. However, statistical significance was not attained for the overall reduction from control. In addition, there were no findings indicative of neurotoxicity at the weekly arena observations or histopathologically and sensory reactivity and grip strength were unaffected. The cause of the reduced activity was unclear but may have been associated with the effects on the gastro-intestinal tract and the decreased body weight gain.

 

There were some findings at the biochemical examination of the blood plasma that suggested a possible effect on liver and kidney function (high bile acid concentration, high alanine amino-transferase activity, low cholesterol concentration and low total protein and albumin concentrations, low creatinine concentration and alterations to plasma electrolytes). There were, however, no histopathological findings in the liver or kidney and these findings were therefore likely due to the metabolism/excretion of the test item and were considered non-adverse.

 

There was no histopathological correlate for the reticulocytosis that occurred in males at all dose levels (there were no microscopic findings in the bone marrow or evidence of extramedullary hematopoiesis) and there was no reduction of hematocrit indicating anemia. This was therefore attributed to normal biological variation.

 

This study included a screen for reproductive/developmental effects and the results obtained were unremarkable. Estrous cycles, pre-coital interval, gestation length and index, mating performance, fertility, litter size, offspring survival to Day 13 of age and sex ratio were not adversely affected by treatment. The number of copulation plugs and sperm counts from vaginal smears at mating were lower than control at 100/75 mg/kg/day, with statistical significance attained for the lower sperm count estimates, but in the absence of any effect on implantation counts or litter size, this was considered non-adverse. Conception rate and fertility index were considered unaffected by treatment.

 

All offspring were macroscopically normal; in particular, no findings were noted on the external genitalia. Ano-genital distances and male nipple counts were not adversely affected by treatment. There was a small reduction of offspring weight gain from Day 1-4 of age, but overall weight gain was considered to be unaffected, therefore this was also non-adverse.

 

The study design also included an assessment of endocrine disruptor relevant endpoints. This objective was met by including the measurement of the hormone thyroxine (T4) in adult males and in offspring at Day 13 of age, by evaluating changes in adult organ weight and gross organ pathology of endocrine-sensitive organs and, because some developmental stages (e.g. gestational and neo-natal) are particularly sensitive to endocrine effects, an external examination of all offspring, measurement of the ano-genital distance of offspring on Day 1 of age and nipple counts for male offspring on Day 13 of age. Although there was a statistically significant increase of thyroxine concentration in parental males receiving 30 or 100/75 mg/kg/day, with the exception of two males receiving 100/75 mg/kg/day which had T4 concentrations that were slightly above the historical control range, all individual values were within the historical control range. Furthermore, there were no significant changes to the absolute and relative weight of the thyroid gland at necropsy, or any histopathological findings in the thyroid gland indicative of a disruption to T4 production (such as follicular cell hypertrophy). It was therefore concluded that, in the context of this study, the test item showed no evidence of being an endocrine disruptor.