N,N-bis(2-hydroxyethyl)-3-[(C16-18)alkoxy]-1-propanamine

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:

22 August 2018 - 20 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Please see materials and methods section.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

Please see materials and methods section.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-hydroxyethyl)-3-[(C16-18)alkoxy]-1-propanamine

EC Number:

930-859-5

Molecular formula:

C23-25H49-53NO3

IUPAC Name:

N,N-bis(2-hydroxyethyl)-3-[(C16-18)alkoxy]-1-propanamine

Test material form:

solid

Remarks:

Amber solid

Specific details on test material used for the study:

Identification :EXP1505486
Physical State/Appearance:Amber colored gelatinous solid
Purity:100%
Batch Number:E00268-015-141
Date Received:02 January 2018
Storage Conditions:Ambient temperature and humidity in darkness; used/formulated in light
Expiry Date:10 June 2019

For the purpose of this study the test item was prepared at the appropriate concentrations in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Covance CRS Research Limited, Shardlow, UK, Analytical Services as part of this study. Results showed the formulations to be stable for at least twenty-one days when stored refrigerated. Formulations were therefore prepared in two batches during the treatment period divided into daily aliquots and stored at approximately 4 ºC in the dark prior to use.
Samples of each test item formulation were taken and analyzed for concentration of EXP1505486 at Covance CRS Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 94-101% of the nominal concentration confirming the accuracy and suitability of the formulation procedure for the purpose of this study

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Covance RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight days during which time their health status was assessed. A total of sixty animals (thirty males and thirty females) were accepted into the study. At the start of treatment the males weighed 183 to 217g, the females weighed 135 to 167g, and were approximately six to eight weeks old.

The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Covance RMS (UK) Limited., Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Annex 5. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Covance CRS Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. There were no deviations from the target range for temperature and the short term deviations from the target range relative humidity were considered not to have affected the purpose or integrity of the study; see Deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily, for up to twenty-eight consecutive days, by gavage using a rubber catheter attached to a disposable plastic syringe.

Vehicle:

arachis oil

Details on oral exposure:

Control animals were treated in an identical manner with 6 mL/kg of Arachis oil BP. Due to the viscous nature of the test item formulations, the dosage volume was increased to 6 mL/kg in the previous preliminary study and the same dose volume was employed for this study. Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentrations in the test samples were determined by Gas Chromatography (GC) using an external standard technique. The test item gave a chromatoraphic profile consisting of a single peak.

The analytical procedure was sucessfully validated with respect to specificity of chromatoraphic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for test item in Arachis Oil BP formulations at nominal concentrations of 3.75mg/ml and 250mg/ml when stored refrigerated for 21 days.

The mean concentrations of test item in test formulations analyzed for the study were within +/- 10% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

The test item was administered daily by oral gavage.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Recovery control

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Intermediate

Doses / concentrations 5

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

High

Doses / concentrations 6

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Recovery high

No. of animals per sex per dose:

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 100 and 250 mg/kg bw/day.

Control animals:

yes

Details on study design:

The dosages employed on the study were selected, in collaboration with the sponsor, based on available toxicity data including a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Study Number LX98XP). In this preliminary study a dosage of 500 mg/kg bw/day and above was demonstrated to be unsuitable for more long-term investigation of toxicity. At 250 mg/kg bw/day, although effects on body weight gain and food consumption were apparent, there was evidence of improvement for both sexes as the study progressed and the extent of the effects observed were considered to be insufficient to preclude this dosage from further investigation of toxicity. Dosages of 0 (control), 30, 100 and 250 mg/kg bw/day were therefore selected for use in investigation.

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 100 and 250 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone over the same treatment period (Arachis oil BP). Two recovery groups, each of five males and five females, were treated either with the high dose (250 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing during the working week, weekends and public holidays. During the treatment-free period, animals were observed daily. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded prior to dosing on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill and, in the case of recovery group animals, on Days 36 and 43 prior to terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically.

OPHTHALMOSCOPIC EXAMINATION: Not specified


HAEMATOLOGY: Yes
Hematological and blood chemical investigations were performed on all non-recovery test and control group animals at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43. Animals were not fasted prior to sampling.

CLINICAL CHEMISTRY: Yes
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin
Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)
Gamma glutamyltranspeptidase
Triglycerides (Tri)


URINALYSIS: Not specified


NEUROBEHAVIOURAL EXAMINATION: Yes
Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin color
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behavior
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Non-recovery animals were tested on one occasion during the last week of treatment and were randomly allocated to the activity monitors. The tests were performed at approximately at least two hours after dosing, under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each non-recovery animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each non-recovery animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

Immunology: Not specified


OTHER:

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -80 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

Histopatholgy and Organ weights were also measured.

Sacrifice and pathology:

Necropsy
On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Histopathology: Please see tissues listed in materials and methods section.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). As the Study Pathologist was part of the Test Facility, a review of the histopathology results for the study was conducted by an appointed Responsible Scientist (V Mowat) working at a designated Test Site (Covance CRS Limited, Huntingdon, Cambridgeshire). A complete histopathology phase report is presented in Annex 1 and this represent the consensus view of both pathologists.

Statistics:

Please see materials and methods section for statistics.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs observed during the treatment period for animals receiving 250 mg/kg bw/day did not indicate any adverse effect of treatment (see Justification of No Observed Adverse Effect Level).
No clinical signs were apparent during the treatment period for animals at 30 or 100 mg/kg bw/day.
No clinical signs were apparent for animals previously treated at 250 mg/kg bw/day during the two-week treatment free recovery period.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For males at 250 mg/kg bw/day, mean body weight gain was statistically significantly lower than control throughout the four week treatment period leading to statistically significantly lower body weight by the end of the treatment period. There was no adverse effect on body weight gain during the recovery period although overall body weight gain from the start of treatment remained statistically significantly lower than control at the end of the recovery period (see Justification of No Observed Adverse Effect Level).
For females at 250 mg/kg bw/day, there were no statistically significant differences from control for body weight gain during the four week treatment period or during the two week treatment-free recovery period.
There were no statistically significant differences from control for body weight gain of either sex at 30 or 100 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For males at 250 mg/kg bw/day, mean food consumption was lower than control throughout the four week treatment period. Following the cessation of treatment, mean food intake for males previously receiving 250 mg/kg bw/day was considered to be similar to control.
For females at 250 mg/kg bw/day, food consumption was considered to be similar to control throughout the four week treatment period and the two week treatment free recovery period.
Mean food consumption for either sex at 30 or 100 mg/kg bw/day was considered to be similar to control throughout the treatment period.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

For males at 250 mg/kg bw/day, food conversion efficiency was lower than control throughout the four week treatment period. There was no adverse effect on food conversion efficiency during the recovery period (see Justification of No Observed Adverse Effect Level).
For females at 250 mg/kg bw/day, food conversion efficiency was considered to be similar to control throughout the four week treatment period and the two week treatment free recovery period.
Food conversion efficiency for either sex at 30 or 100 mg/kg bw/day was considered to be similar to control throughout the treatment period.

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

effects observed, treatment-related

Description (incidence and severity):

At 250 mg/kg bw/day, mean water consumption for both sexes assessed during Week 3 of the study was generally higher than control.
For males at 100 mg/kg bw/day, mean water consumption during Week 3 tended to be lower than control, but this was considered to be unrelated to treatment (see Justification of No Observed Adverse Effect Level). Mean water consumption for females at 100 mg/kg bw/day and both sexes at 30 mg/kg bw/day was considered to be similar to control during Week 3.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no adverse effect of treatment on hematology parameters at the end of the four week treatment period for either sex 30, 100 or 250 mg/kg bw/day (see Justification of No Observed Adverse Effect Level).
For animals previously treated at 250 mg/kg bw/day, occasional statistically significant differences from control for hematology parameters at the end of the two week treatment-free recovery were considered to be of no toxicological significance (see Justification of No Observed Adverse Effect Level).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

For males at 250 mg/kg bw/day, mean total protein and cholesterol levels were statistically significantly lower than control at the end of the treatment period. Mean albumin/globulin (A/G) ratio was statistically significantly higher than control for these treated males at the end of the treatment period, however, all individual values for albumin/globulin ratio were within the historical control range and this finding was considered not to represent an adverse effect of treatment.
Statistically significant differences for other blood chemistry parameters for these males at the end of the treatment period were considered to be incidental and of no toxicological significance (see Justification of No Observed Adverse Effect Level).
There was considered to be no adverse effect of treatment on blood chemistry parameters at the end of the four week treatment period for either sex at 30 or 100 or for females at 250 mg/kg bw/day (see Justification of No Observed Adverse Effect Level).
For animals previously treated at 250 mg/kg bw/day, statistically significant differences for blood chemistry parameters at the end of the recovery period were considered to be incidental and of no toxicological significance (see Justification of No Observed Adverse Effect Level).

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioral Assessments
Intergroup differences in behavioral assessments did not indicate any effect of treatment for either sex at 30, 100 or 250 mg/kg bw/day.
Functional Performance Tests
Intergroup differences in forelimb or hindlimb grip strength did not indicate any effect of treatment for either sex at 30, 100 or 250 mg/kg bw/day (see Justification of No Observed Adverse Effect Level).
Sensory Reactivity Assessments
Intergroup differences in sensory reactivity assessments did not indicate any effect of treatment for either sex at 30, 100 or 250 mg/kg bw/day.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Mean absolute and body weight relative liver weights were statistically significantly higher than control for females at 100 mg/kg bw/day and both sexes at 250 mg/kg bw/day at the end of the treatment period. Mean absolute and body weight relative liver weights were also statistically significantly higher than control for females at the end of the two week recovery period (see Justification of No Observed Adverse Effect Level). The increases in liver weights at the end of the treatment period were not accompanied by any macroscopic necropsy findings or evidence of histopathological change and most likely reflect adaptive changes in liver metabolism. These findings were, therefore, considered not to represent an adverse effect of treatment.
Mean absolute and body weight relative kidney weights were statistically significantly higher than control for males at all dosages at the end of the treatment period, although group mean values showed no consistent dosage relationship (see Justification of No Observed Adverse Effect Level). There were no macroscopic necropsy findings for the male kidneys at the end of treatment period and, in the absence of any or evidence of histopathological change, this finding was considered not to represent an adverse effect.
Other statistically significant differences from control for absolute and body weight relative organ weights observed during the study were considered to be incidental and of no toxicological significance (see Justification of No Observed Adverse Effect Level).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 250 mg/kg bw/day, four males showed small prostate and seminal vesicles at the end of the treatment period.
Necropsy finding for females at 100 or 250 mg/kg bw/day at the end of the treatment period were considered to be incidental and of no toxicological significance (see Justification of No Observed Adverse Effect Level). No necropsy findings were apparent for either sex at 30 mg/kg bw/day or for males at 100 mg/kg bw/day at the end of the treatment period.
Necropsy finding for females previously treated at 250 mg/kg bw/day at the end of the recovery period were considered to be incidental and of no toxicological significance (see Justification of No Observed Adverse Effect Level).

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Small Intestine
Foamy macrophage aggregates were noted in the mucosa of the jejunum at a mild severity for 2/5 males and 3/5 females and at a moderate severity for 3/5 males and 2/5 females in Group 4. They were also present at a minimal severity in 3/5 males and 5/5 females and at a mild severity for 2/5 males in Group 3. Foamy macrophages were present in 1/5 Group 2 males at a minimal severity.
Foamy macrophage aggregates were noted in the mucosa of the duodenum at a minimal severity for 2/5 males and 4/5 females, and at a mild severity for 1/5 males in Group 4.
Foamy macrophage aggregates were noted in the mucosa of the ileum at a minimal severity for 2/5 males and at a mild severity for 1/5 males in Group 4.
After a two week treatment free period there was evidence of reversibility but recovery was incomplete in the jejunum.

Mesenteric Lymph Node
Foamy macrophage aggregates were noted in the mesenteric lymph node at a moderate severity for all (5/5) females and at a moderate severity for all (4/4) males examined in Group 4. They were also present at a mild severity in all (5/5) females and at a moderate severity for all (5/5) males in Group 3. Foamy macrophages were present at a minimal severity in 2/5 males and 2/5 females in Group 2.
After a two week treatment free period there was evidence of reversibility with a reduction in severity in some animals, but recovery was incomplete.

Male Reproductive Tract
Decreased secretion was present in the seminal vesicles of 4/5 Group 4 males with decreased secretion in the prostrate of 1/5 Group 4 males. Decreased secretion was present in 2/5 Group 3 males correlating with a reduction in weight.
After a two week treatment free period there was good evidence of reversibility, but recovery was incomplete in one animal, correlating with a low organ weight.

Histopathological findings: neoplastic:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

food efficiency

gross pathology

histopathology: non-neoplastic

water consumption and compound intake

Remarks on result:

other: NOAEL of at least 100 mg/kg bw/day should be considered when assessing potential health effects of the test item for man.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

intestine

mesenteric lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Treatment of males at 250 mg/kg bw/day was associated with lower body weight gain, food consumption and food conversion efficiency, compared to control, throughout the treatment period. Following the cessation of treatment, there was no subsequent adverse effects on body weight gain, food consumption or food conversion efficiency, although overall body weight from the start of treatment remained lower than control at termination. For females at this dosage, no adverse effects of treatment on body weight gain, food consumption or food conversion efficiency were apparent during the treatment or recovery periods. Despite the obvious effects on body weight and food consumption for males during the treatment period, clinical signs for both sexes at 250 mg/kg bw/day, were generally restricted to incidences of increased post-dosing salivation during the treatment period, a finding considered to reflect slight distaste or irritancy of the test item rather than a systemic effect of treatment. Mean water consumption was generally higher than control for both sexes at this dosage; this finding may also reflect slight irritancy of the test item, although it could also have been influenced by disturbance of the lower intestines at this dosage. 

There was considered to be no effect of treatment on clinical signs, body weight gain, food consumption, food conversion efficiency or water consumption for either sex at 30 or 100 mg/kg bw/day.   

Performance of the animals during behavioral assessments, functional performance tests and sensory reactivity assessments did not indicate any neurological effect of the Test Item at 30, 100 or 250 mg/kg bw/day. 

For males at 250 mg/kg bw/day, mean cholesterol and total protein levels were statistically significantly lower than control at the end of the treatment period, with the majority of individual values for these treated animals being lower than the respective historical control range. Mean albumin/globulin (A/G) ratio were higher than control at the end of the treatment period but all individual values for these treated animals were within the historical control range. These blood chemistry parameters may have been influenced by altered hepatic metabolism, as indicated by increased liver weight, or altered absorption from the small intestines as a result of histopathological changes, although similar changes for the liver and small intestines were apparent for females at this dosage without any accompanying effect on these blood chemistry parameters. 

Other statistically significant differences for blood chemistry or hematology parameters for animals receiving 30, 100 or 250 mg/kg bw/day were considered to be incidental and of no toxicological significance.

For females at 100 mg/kg bw/day and both sexes at 250 mg/kg bw/day, mean liver weights were statistically significantly higher than control at the end of the treatment period. Body weight relative liver weights are generally considered to be the better indicator of toxicological effect and all, or the majority of individual values for these treated animals exceeded the historical control range. Higher liver weights persisted for females at 250 mg/kg bw/day to the end of the recovery period, with the majority of individual relative liver weights exceeding the historical control range. The increases in liver weights at the end of the treatment period were not accompanied by any evidence of histopathological change and most likely reflect adaptive changes in liver metabolism. These findings were, therefore, considered not to represent an adverse effect of treatment. 

Higher kidney weights were apparent for males at all dosages at the end of the treatment period but group mean values showed no consistent dosage relationship. There was no similar increase in kidneys weights apparent for females and the increases in kidneys weights for males were no longer apparent at the end of the recovery period. There were no macroscopic necropsy findings or evidence of histopathological change for the male kidneys at the end of treatment period and, while the cause of this increase in kidney weights was unclear, it was considered not to represent an adverse effect. Other statistically significant differences from control for absolute and body weight relative organ weights observed during the study were considered to be incidental and of no toxicological significance (see Justification of No Observed Adverse Effect Level). 

At 250 mg/kg bw/day, four males showed small prostate and seminal vesicles at necropsy at the end of the treatment period. Although mean absolute and body weight relative values were lower than control, differences failed to attain statistical significance and relative values showed no clear dosage relationship. However, histopathology examination revealed decreased secretion in the seminal vesicles of 4/5 males and decreased secretion in the prostrate of 1/5 males at 250 mg/kg bw/day at the end of the treatment period. Decreased secretion was also present at the end of treatment for 2/5 males at 100 mg/kg bw/day. After the two-week treatment free recovery period at 250 mg/kg bw/day there was evidence of reversibility with a reduction in severity in some animals, but recovery was incomplete. The decreased secretion in the seminal vesicles at 250 mg/kg bw/day may be due to a direct effect on the male reproductive tract or may have been influenced by the reduction in body weight at this dosage and is considered to be potentially adverse. 

Other macroscopic necropsy findings observed during the study were considered to be incidental and of no toxicological significance. 

Histopathological examination of the small intestines revealed foamy macrophage aggregates in the mucosa of the jejunum (mild or moderate severity) and the duodenum (minimal or mild severity) for both sexes and, additionally, the ileum (minimal or moderate severity) for males at 250 mg/kg bw/day at the end of the treatment period. Following the two-week treatment free recovery period there was evidence of reversibility at 250 mg/kg bw/day but recovery was incomplete in the jejunum. Foamy macrophage aggregates were also observed in the mucosa of the jejunum at a minimal or mild severity for males and a minimal level for females at 100 mg/kg bw/day at the end of the treatment period. At 30 mg/kg bw/day, foamy macrophages in the mucosa of the jejunum were restricted to one male at minimal severity, at the end of the treatment period.

Histopathological examination also revealed foamy macrophage aggregates in the mesenteric lymph node (moderate or marked severity) of both sexes at 250 mg/kg bw/day. Following the two-week treatment free recovery period there was evidence of a reduction in severity in some animals, but recovery was incomplete. Foamy macrophage aggregates were also observed, at a mild or moderate severity, for both sexes at 100 mg/kg bw/day at the end of treatment. Foamy macrophage aggregates were observed for two males and two females at 30 mg/kg bw/day, but only at a minimal severity, at the end of treatment.

The foamy macrophage aggregates in the small intestine and mesenteric lymph node are considered likely to be related to the absorption of the test item and/or metabolites and are considered to represent the most significant findings on the study. At 250 mg/kg bw/day, all three areas of the small intestine were consistently affected, and this may be the underlying cause for the decreased body weight gain for males at this dosage. At the severity levels of moderate or marked observed for males at 100 mg/kg bw/day and both sexes at 250 mg/kg bw/day, the function of the mesenteric lymph node may be compromised and this is considered to indicate an adverse effect. At 30 mg/kg bw/day, these findings were restricted to a few animals at a minimal severity and this dosage is, therefore, regarded as being the No Observed Adverse Effect Level (NOAEL) for toxicity.

Whilst, the histopathological findings for the small intestines and mesenteric lymph nodes observed on this study are considered to represent an adverse histopathological finding, their significance when assessing health effects for man are more equivocal, particularly at 100 mg/kg bw/day, where they were not supported by any other findings considered to be of toxicological significance. The restriction of histopathological findings to macrophages within the small intestines and associated lymph nodes suggests that these findings resulted from direct contact within the intestinal tract and the proliferation of macrophages may represent an adaptive response to the presence of the test item within the small intestines. The persistence of these macrophages probably reflects an inability to degrade the phagocytosed material. The more pronounced effects in the jejunum are consistent with the comparatively longer transient time under normal physiological conditions, compared to the duodenum and ileum, resulting in longer exposure to the test item. The test item is known to be a weak skin corrosive (1C) and, although no histopathological findings indicative of irritation were apparent for the intestinal tissues per se, the slightly corrosive nature of the test item may have been a contributing factor in the observed reaction to the presence of the test item within the small intestines. It should be noted that oral studies with certain mineral oils and waxes have also resulted in macrophages displaying similar vacuolated “foamy” cytoplasm. A review and workshop reported that macrophage proliferations in lymph nodes (described as micro-granulomas), where these macrophages may display vacuolated “foamy” cytoplasm is seen with oral ingestion of mineral oils and waxes (Carlton., W.W.et.al.). Within this review there was consideration as to whether the potential differences between rodents and humans in the metabolic utilization of such products may mislead the risk and relevance of these findings to man from exposure to these materials.

As previously indicated the proliferative macrophage response is potentially a natural reaction involving the adaptive macrophage reaction to a foreign material that has been absorbed across the epithelial boundary within the gut lumen. As adaptive responses can be interpreted as a “Discriminating Factor” in the recognition and classification of adverse/non adverse findings (ECETOC Technical Report Number 85, 2002), it is possible that the findings observed for the macrophages within this study can be included within such as category. The macrophage response may be regarded as a non-adverse response or not indicative of a true systemic toxic response and the observed changes were not associated with any degenerative change. Additionally these findings, at 250 mg/kg bw/day, showed evidence of recovery over the two week recovery period; whilst full recovery was not apparent for the jejunum at the end of the recovery phase, there was no reason to believe that these findings would not have been fully resolve over a more extended period. It is considered that the presence of foamy macrophages in the small intestine and mesenteric lymph node within this study does not meet the criteria set within the United Nations’ Globally Harmonised System (GHS) for classification (The Classification, Labelling and Packaging (CLP) Regulation (EC) No. 1272/2008, Annex I: 3.9.2.7.3) as it does not represent significant organ damage, multi-focal or diffuse necrosis, fibrosis or granuloma formation in vital organs with regenerative capacity, morphological changes (reversible or not) resulting in marked organ dysfunction nor appreciable cell death in vital organs incapable of regeneration. The macrophage findings observed on this study may, however, fall within the criteria considered not to support classification as they could be considered to represent adaptive responses that are not toxicologically relevant ((CLP) Regulation (EC) No. 1272/2008, Annex I: 3.9.2.8.1).

Overall, based on the results of this study, the clear No Observed Adverse Effect Level (NOAEL) for toxicity for the rat was considered to be 30 mg/kg bw/day, principally due to the presence of foamy macrophages within the small intestines and mesenteric lymph nodes. However, due to the potentially adaptive nature of the macrophage findings observed, a NOAEL of at least 100 mg/kg bw/day should be considered when assessing potential health effects of the test item for man.

JUSTIFICATION OF NO OBSERVED ADVERSE EFFECT LEVEL

Oral administration of the test item to rats for a period of twenty-eight consecutive days at dose levels of up to 250 mg/kg bw/day resulted in treatment-related changes. The ‘No Observed Adverse Effect Level’ (NOAEL) was, therefore considered to be 30 mg/kg bw/day.

The following differences between treated and control animals were considered not to be indicative of test item toxicity:

Clinical Observations

·                At 250 mg/kg bw/day, incidences of increased post-dosing salivation were apparent, to some extent, for all animals during the treatment period, with this clinical sign first being apparent for males from Day 5 and for females from Day 6 of dosing. Increased post-dosing salivation is often observed when animals are dosed via the oral gavage route and is generally considered to reflect slight distaste or irritancy of the test item rather than a systemic effect of treatment.

·                At 250 mg/kg bw/day, noisy respiration was apparent for one female on Day 8 of the study; this finding was considered to be incidental and likely to reflect difficulties in dosing the animal on this isolated occasion. 

Functional Performance Tests

·                At 30 mg/kg bw/day, hindlimb grip strength during the first trial for females was statistically significantly lower than control.  No statistically significant differences were apparent for these females during the next two trials of hindlimb grip strength or during the assessments of forelimb grip strength. In the absence of any similar effect on grip strength at higher dosages, this isolated finding was clearly incidental and unrelated to treatment.

Body weight

·                At 250 mg/kg bw/day, following the cessation of treatment, body weight gain for males during the first week of the recovery period was statistically significantly higher than control during the first week of the recovery period. This finding was considered to reflect initial recovery from the lower body weight gain observed for these animals during the treatment period and, as such, was considered not to represent an adverse effect.

Food Conversion Efficiency

·                At 250 mg/kg bw/day, following the cessation of treatment, food conversion efficiency for males during the first week of the recovery period was superior to control during the first week of the recovery period. This finding was considered to reflect the improvement in body weight gain and food consumption for these males following the withdrawal of treatment and, as such, was considered not to represent an adverse effect.

Water Consumption

·                At 100 mg/kg bw/day, mean water consumption of males during the period of gravimetric assessment (Week 3) tended to be lower than control but there was no consistent pattern for water intake of females at this dosage. At the higher dosage of 250 mg/kg bw/day, treatment had been associated with higher water intake for both sexes. An association with treatment for the lower water consumption for males at 100 mg/kg bw/day, therefore, appears unlikely and this finding was considered not to represent an adverse effect of treatment.

Hematology

·      For males at 250 mg/kg bw/day, mean hemoglobin was statistically significantly lower than control at the end of the treatment period, with three individual values being lower than the historical control range. The mean value for mean corpuscular hemoglobin was also statistically significantly lower than control at the end of the treatment period, but all individual values were within the historical control range. Additionally, for males at all dosages, the mean values for mean corpuscular volume were statistically significantly lower than control at the end of the treatment period, but again all individual values were within the historical control range. For females at 250 mg/kg bw/day, statistically significantly lower mean corpuscular hemoglobin was apparent at the end of treatment, compared to control, with three individual values for these treated females being below the historical control range. Additionally, statistically significantly higher erythrocyte count and lower mean corpuscular volume were also apparent at the end of treatment, compared to control, but the majority of individual values for these treated females were within the respective historical control range. The findings observed for both male and female hematology parameters at the end of the treatment period were not supported by any effect on the number of reticulocytes for either sex and were unconvincing. In the absence of any supporting histopathological change these findings were considered not to represent an adverse effect.

·      For animals previously treated at 250 mg/kg bw/day, mean activated partial thromboplastin time at the end of the recovery period was statistically significantly shorter for males and statistically significantly longer for females, compared to control. One individual female value for these recovery animals exceeded the historical control range, all other individual values for both sexes were within the respective historical control range. Given the contrasting nature of this finding between the two sexes, and the lack of any effect on clotting times at the end of the treatment period, these findings appeared unlikely to reflect previous treatment with the test Item. In view of this, these findings were considered to be incidental and unrelated to treatment.

·      For males previously treated at 250 mg/kg bw/day the mean number of eosinophils was statistically significantly lower than control at the end of the recovery period, but all individual values for these recovery males were within the historical control range. There were no effects on the numbers of eosinophils or other white blood cell types during the treatment period, and this finding, occurring at a time where treatment related histopathological findings were showing at least some evidence of recovery, was considered to be incidental and of no toxicological significance.   

Blood chemistry  

·      For males at 250 mg/kg bw/day, mean albumin/globulin (A/G) ratio was statistically significantly higher than control at the end of the treatment period. Whilst this finding occurred in the presence of lower mean total protein, all individual values for albumin/globulin ratio were within the historical control range and this finding was considered not to represent an adverse effect of treatment.

·      At 250 mg/kg bw/day, the mean triglycerides level for males was statistically significantly lower than control at the end of the treatment period, but, all individual values were within the historical control range. This finding was considered to reflect high control values, as two individual control values exceeded the historical control range, rather than an effect of treatment.

·      For males at 250 mg/kg bw/day, the mean bilirubin level was statistically significantly lower than control at the end of the treatment period, but all individual values were within the historical control range. This finding was considered to reflect high control values, as two individual control values exceeded the historical control range, rather than an effect of treatment.

·      For both sexes previously treated at 250 mg/kg bw/day, mean alkaline phosphatase activity was statistically significantly lower than control at the end of the recovery period but the majority of the individual values for these recovery animals were within the historical control range. This intracellular enzyme is usually contained within the cells and is only present in the plasma due to cell turnover, leakage and cell death. Whilst an increase in this enzyme may indicate damage to the organ containing it, a decrease is considered to be of no toxicological significance. This finding was, therefore, considered to be incidental and unrelated to previous treatment with the test item.

·      For males previously treated at 250 mg/kg bw/day, the mean level of albumin and mean albumin/globulin (A/G) ratio were statistically significantly higher than control at the end of the recovery period. The finding for albumin contrast with that observed at the end of the treatment period when the mean value at this dosage was lower than control. With the exception of one individual value for albumin/globulin ratio, all individual values for these parameters were within the respective historical control range and these findings were considered to be of no toxicological significance.

·      For males previously treated at 250 mg/kg bw/day, the mean cholesterol level was statistically significantly lower than control at the end of the recovery period. Whilst, this finding was consistent with that observed for males at the end of the treatment period at this dosage, all individual values for these recovery males were within the historical control range and this finding was considered not to represent an adverse effect.

·      For males previously treated at 250 mg/kg bw/day, statistically significantly higher chloride and statistically significantly lower triglyceride levels, compared to control, were apparent at the end of the recovery period. For females previously treated at 250 mg/kg bw/day, statistically significantly higher glucose and statistically significantly lower phosphorus levels, compared to control, were apparent at the end of the recovery period. Values for these parameters for the affected treated animals were within the historical control range. In the absence of any similar findings considered to be related to treatment for these parameters at the end of the treatment period, these findings for these recovery animals at the end of the study were considered to be incidental and unrelated to previous treatment with the test item.

Necropsy  

·      One female at 100 mg/kg bw/day and one female at 250 mg/kg bw/day showed a small uterus and cervix at necropsy at the end of the treatment period. There were no statistically significant differences apparent for mean uterus weights during the study. No evidence of histopathological change was apparent for the female at 100 mg/kg bw/day. Uterine atrophy and anestrus were observed for the female at 250 mg/kg bw/day but, due to the low incidence of this finding, it was considered to be incidental and unrelated to treatment.

·      At 100 mg/kg bw/day, one female showed small kidneys at the end of the treatment period. At 250 mg/kg bw/day, one female showed small kidneys and another female showed a fluid filled right kidney with increased pelvic space at the end of the treatment period. A further female previously treated at 250 mg/kg bw/day showed increased pelvic space within the left kidney at the end of the recovery period. No statistically significant differences from control for kidney weights were apparent for females during the study and these was no supporting histopathological change in the kidneys for females at 250 mg/kg bw/day at the end of treatment period. In isolation, these findings were considered to be incidental and unrelated to treatment.

·      One male, previously treated at 250 mg/kg bw/day, showed a small left adrenal at necropsy at the end of the recovery period. In the absence of any similar findings at the end of the treatment period, this isolated finding was considered to be incidental and unrelated to previous treatment with the test item.

Organ weights

·      Statistically significantly higher liver weights, compared to control, were apparent for females at 100 mg/kg bw/day and both sexes at 250 mg/kg bw/day at the end of the treatment period. Body weight relative liver weights are generally considered to be the better indicator of toxicological effect and all, or the majority of individual values for these treated animals exceeded the historical control range. Higher liver weights persisted for females at 250 mg/kg bw/day to the end of the recovery period with the majority of individual relative liver weights exceeding the historical control range. The increases in liver weights at the end of the treatment period were not accompanied by any macroscopic necropsy findings or evidence of histopathological change and most likely reflect adaptive changes in liver metabolism. These findings were, therefore, considered not to represent an adverse effect of treatment.

·      Statistically significantly higher kidney weights were apparent for males at all dosages at the end of the treatment period. Body weight relative weights are generally considered to be the better indicator of toxicological effect for this organ and 3/5, 2/5 and 4/5 individual relative values exceeded the historical control range at 30, 100 and 250 mg/kg bw/day respectively. Whilst, the highest relative mean value occurred at 250 mg/kg bw/day there was a lack of dosage relationship at lower dosages. No similar increase in mean kidneys weights were apparent for females at the end of treatment and the increases in kidneys weights for males were no longer apparent by the end of the recovery period. There were no macroscopic necropsy findings for the male kidneys at the end of treatment period and, in the absence of any or evidence of histopathological change, this finding was considered not to represent an adverse effect.

·      For females at 100 mg/kg bw/day, mean ovary weights were higher than control at the end of the treatment period, principally due to two females whose absolute and body weight-relative ovary weights exceeded the respective historical control range. In the absence of any similar increase in weights or evidence of histopathological change for the ovaries of females at 250 mg/kg bw/day at the end of the treatment period, this finding at 100 mg/kg bw/day was considered to be incidental and unrelated to treatment.

·      For females at 30 mg/kg bw/day, mean pituitary weights were higher than control at the end of the treatment period with the majority of the individual absolute and body weight relative values exceeding the respective historical control range. There were no similar increases for pituitary weights for females at 100 or 250 mg/g bw/day or evidence of histopathological change for females at 250 mg/kg bw/day at the end of the treatment period. This finding at 30 mg/kg bw/day was, therefore, considered to be incidental and unrelated to treatment. 

Histopathology

·      One female at 250 mg/kg bw/day showed uterine atrophy and anestrus at the end of the treatment period but, due to the low incidence, this finding was considered to be incidental and unrelated to treatment. This female also showed hyperplasia of the non-glandular mucosa in the stomach; this finding wasn’t apparent for other treated animals and, due to the low incidence, this finding was considered to be incidental and unrelated to treatment.

Applicant's summary and conclusion

Conclusions:

Overall, based on the results of this study, the clear No Observed Adverse Effect Level (NOAEL) for toxicity for the rat was considered to be 30 mg/kg bw/day, principally due to the presence of foamy macrophages within the small intestines and mesenteric lymph nodes. However, due to the potentially adaptive nature of the macrophage findings observed, a NOAEL of at least 100 mg/kg bw/day should be considered when assessing potential health effects of the test item for man.

Executive summary:

Introduction

The study was designed to establish the effects of repeated oral administration of the test item to rats over a period of twenty-eight consecutive days. It also assessed the ability of the animals to recover from any toxicity following the withdrawal of treatment. The study is compatible with the following regulatory guidelines:

·        Commission Directive 96/54/EC (Method B7).

·        The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 100 and 250 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone over the same treatment period (Arachis oil BP). Two recovery groups, each of five males and five females, were treated either with the high dose (250 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

All animals were subjected to gross necropsy examination and histopathological examination of selected tissues was performed.

Results

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

There were no clinical signs observed during the study that were considered to represent an adverse systemic effect of treatment at 30, 100 or 250 mg/kg bw/day.

Behavioral Assessment

Behavioral assessments did not indicate any adverse effect of treatment for either sex at 30, 100 or 250 mg/kg bw/day.

Functional Performance Tests

Results from functional performance tests did not indicate any effect of treatment for either sex at 30, 100 or 250 mg/kg bw/day.

Sensory Reactivity Assessments

Sensory reactivity assessments did not indicate any adverse effect of treatment for either sex at 30, 100 or 250 mg/kg bw/day.

Body Weight

At 250 mg/kg bw/day, mean body weight gain for males was statistically significantly lower than control throughout the treatment period leading to statistically significantly lower body weight by the end of the treatment. Following the cessation of treatment, body weight gain for males was statistically significantly higher than control during the first week of the recovery period, however, mean body weight at the end of the recovery period remained statistically significantly lower than control. 

Body weight gain for either sex at 30 or 100 mg/kg bw/day and for females at 250 mg/kg bw/day appeared unaffected by treatment.

Food Consumption

At 250 mg/kg bw/day, mean food consumption for males was lower than control throughout the treatment period. For females at this dosage, mean food consumption was considered to be unaffected by treatment during the dosing period. Food intake was similar to control for both sexes during the recovery period.   

Food consumption for either sex at 30 or 100 mg/kg bw/day appeared unaffected by treatment.

Food Conversion Efficiency

At 250 mg/kg bw/day, food conversion efficiency for males was lower than control throughout the treatment period. For females at this dosage, food conversion efficiency was considered to be unaffected by treatment during the dosing period. Food conversion efficiency for either sex was not adversely affected by previous treatment during the recovery period.

Food conversion efficiency for either sex at 30 or 100 mg/kg bw/day appeared unaffected by treatment.

Water Consumption

At 250 mg/kg bw/day, mean water consumption for both sexes assessed during Week 3 of the study was generally higher than control.  

At 100 or 30 mg/kg bw/day, observed differences in water consumption during Week 3 were considered to be incidental and unrelated to treatment.

Hematology

Assessment of hematology parameters at 30, 100 or 250 mg/kg bw/day at the end of treatment and at 250 mg/kg bw/day at the end of the two week treatment-free recovery period did not indicate any adverse effect of treatment.

Blood Chemistry

At 250 mg/kg bw/day, mean cholesterol and total protein were statistically significantly lower than control at the end of treatment period.

Assessment of other blood chemistry parameters at 30, 100 or 250 mg/kg bw/day at the end of treatment and at 250 mg/kg bw/day at the end of the two week treatment-free recovery period did not indicate adverse effect of treatment.

Necropsy

At 250 mg/kg bw/day, four males showed small prostate and seminal vesicles at the end of the treatment period.

The type, incidence and distribution of other macroscopic necropsy findings at 30, 100 or 250 mg/kg bw/day at the end of treatment and at 250 mg/kg bw/day at the end of the two week treatment-free recovery period did not indicate any adverse effect of treatment.

Organ Weights

Mean absolute and body weight relative liver weights were statistically significantly higher than control at the end of the treatment period for females at 100 mg/kg bw/day and both sexes at 250 mg/kg bw/day. At 250 mg/kg bw/day, the higher mean liver weights persisted for females until the end of the two week recovery period. 

For males at all dosages, mean absolute and body weight relative kidney weights at the end of the treatment period were statistically significantly higher than control but mean values showed no consistent dosage relationship.

Histopathology

Small Intestine

At 250 mg/kg bw/day, foamy macrophage aggregates were noted in the mucosa of the jejunum (mild or moderate severity) for all animals, in the mucosa of the duodenum (minimal or mild severity) for most animals and in the mucosa of the ileum (minimal or mild severity) of most males at the end of the treatment period. After a two week treatment free period there was evidence of reversibility but recovery was incomplete in the jejunum.

At 100 mg/kg bw/day, foamy macrophage aggregates were noted in the mucosa of the jejunum for all males (minimal or mild severity) and all females (minimal severity) at the end of the treatment period.

At 30 mg/kg bw/day, foamy macrophage aggregates in the mucosa of the jejunum were only present for one male at a minimal severity at the end of the treatment period.

Mesenteric Lymph Node

At 250 mg/kg bw/day, foamy macrophage aggregates were noted in the mesenteric lymph node of all animals at moderate or marked severity at the end of the treatment period. After a two week treatment free period there was evidence of reversibility with a reduction in severity in some animals, but recovery was incomplete.

At 100 mg/kg bw/day, foamy macrophage aggregates were noted in the mesenteric lymph node of all animals at mild or moderate severity at the end of the treatment period.

At 30 mg/kg bw/day, foamy macrophage aggregates were only noted in the mesenteric lymph node of two males and two females at a minimal severity at the end of the treatment period.

Male Reproductive Tract

At 250 mg/kg bw/day, decreased secretion was present in the seminal vesicles of four males with decreased secretion also being observed in the prostrate of one male at the end of the treatment period. After a two week treatment free period there was good evidence of reversibility, but recovery was incomplete for the seminal vesicles in one male.

At 100 mg/kg bw/day, decreased secretion was present in the seminal vesicles of two males at the end of the treatment period.

Conclusion

Overall, based on the results of this study, the clear No Observed Adverse Effect Level (NOAEL) for toxicity for the rat was considered to be 30 mg/kg bw/day, principally due to the presence of foamy macrophages within the small intestines and mesenteric lymph nodes. However, due to the potentially adaptive nature of the macrophage findings observed, a NOAEL of at least 100 mg/kg bw/day should be considered when assessing potential health effects of the test item for man.