Registration Dossier

Administrative data

Description of key information

OECD 422: NOAEL (males) = 100 mg/kg bw/d
OECD 408: NOAEL (males/females) = 300 mg/kg bw/d
OECD 443: NOAEL (males/females) = 100 mg/kg bw/d

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 January 2014 - 28 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Remarks:
Two animals were incorrectly identified resulting in the animals being dosed for two days based on the incorrect body weights.
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Monitoring authority, Dept. of Health UK, 2014
Limit test:
no
Species:
rat
Strain:
Wistar
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 Harlan Laboratories U.K. Ltd., Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for five days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 161 to 192g, the females weighed 141 to 174g, and were approximately six to eight weeks old.

The animals were housed in groups of three or four 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, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Appendix 23. 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 Harlan Laboratories Ltd., 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. Short term deviations from these targets 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.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
Test item preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results from the previous study show the formulations to be stable for at least twenty one days. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.

Samples of each test item formulation were taken and analyzed for concentration of AS1100 at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 7% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by gas chromatography using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.
The formulations investigated were found to comprise test item in the range of 97 to 107 % and thus the required content limit with reference to the nominal content was met.
The test item was found to be stable in the formulations when kept for 21 days in the refrigerator (4 °C) due to results which met the variation limit of 10 % from the time-zero mean.
The results indicate the accurate use of the test item and vehicle during the study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Duration of treatment / exposure:
Ninety consecutive days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
10, 100 and 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
Dose levels were selected in collaboration with the Sponsor based on previous toxicity information from a study of a significantly shorter duration which indicated adverse stomach and liver findings at 300 and 1000 mg/kg bw/day, where the incidence and severity of findings were dose-related and the No Observed Adverse Effect Level (NOAEL) assigned in this OECD 422 study was 300 mg/kg bw/day. Therefore, 300 mg/kg bw/day was selected as a high dose level due to the potential for the prolonged exposure period to exacerbate any similar effects.
The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.

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.
Observations and examinations performed and frequency:
Clinical Observations
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 throughout the treatment period. All observations were recorded.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each 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.


Functional Performance Tests
Motor Activity.
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (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 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 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.


Body weight
Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.


Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.


Ophthalmoscopic Examination
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

Laboratory Investigations
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.

Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
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++)
Sacrifice and pathology:
Pathology
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.

All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus, Liver.

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:

Adrenals, Ovaries Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint)•, Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Rectum, Caecum, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides ♦, Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes*, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Testes ♦, Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Tongue• Lungs (with bronchi) # Trachea, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary glands, Uterus (with cervix), Muscle (skeletal)•, Vagina.
* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative
• Retained only and not processed

All tissues were dispatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). All tissues from control and 300 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.

Microscopic examination was conducted by the Study Pathologist (Peter Millar at Peter Millar Associates Ltd., 3 Queen Charlotte Lane, Edinburgh, EH6 6AY).
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p>0.05 (not significant)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See results
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
There were no unscheduled deaths.


Clinical Observations
Neither the type, incidence or distribution of clinical signs apparent indicated an adverse effect of treatment at 10, 100 or 300 mg/kg bw/day.

Animals of either sex treated with 300 mg/kg bw/day had increased salivation post-dose from Day 22 (Females) and Day 23 (Males). Eight males and six females treated with 100 mg/kg bw/day also showed incidences of increased salivation from Day 44 (Females) and Day 45 (Males). Observations of this nature are commonly experienced following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be of toxicological importance.

No such effects were detected in animals of either sex treated with 10 mg/kg bw/day.

Functional Observations
Behavioral Assessments
There were no treatment-related changes in the behavioural parameters measured.

All inter and intra group differences in urination and defecation were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.

Functional Performance Tests
There were no toxicologically significant changes in functional performance.

Males treated with 10 mg/kg bw/day had a statsitically significant reduction in overall activity. No dose relationship was apparent and in the absence of any associated supporting clinical observations to suggest a neurotoxic effect of treatment, this intergroup difference was considered not to be toxicologically significant.

Females treated with 100 mg/kg bw/day had a statistically significant reduction in overall mobility. No dose relationship was evident, therefore the intergroup differences were considered to represent normal biological variation rather than an effect of treatment.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.

All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.


Body Weight
Overall body weight gain was lower than control for males at 300 mg/kg bw/day. Group mean body weight gains were generally lower than control throughout the treatment period with differences from control attaining statistical significance during Weeks 7, 10 and 11. Whilst an effect of treatment on body weight development was apparent in males at this dose level, the magnitude of the effect observed was considered insufficient in severity to consider the effect adverse.

No effects of treatment on body weight development were apparent for females treated with 300 mg/kg bw/day or animals of either sex treated with 10 or 100 mg/kg bw/day.


Food Consumption
There was no obvious effect of treatment on food consumption at 10, 100 or 300 mg/kg bw/day.


Water Consumption
Daily visual inspection of water bottles did not reveal any overt differences in water consumption at 10, 100 or 300 mg/kg bw/day.

Ophthalmscopic Examination
There were no treatment-related ocular effects.

Laboratory Investigations
Hematology
Assessment of hematology parameters did not indicate any obvious effect of treatment at 10, 100 or 300 mg/kg bw/day.

Males treated with 300 mg/kg bw/day showed a statistically significant reduction in mean corpuscular hemoglobin concentration and a statistically significant increase in hematocrit, neutrophil count and platelet count. Males treated with 100 mg/kg bw/day also showed a statistically significant increase in platelet count. All treated females showed a statistically significant reduction in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. Females treated with 300 and 100 mg/kg bw/day showed a statistically significant reduction in mean corpuscular volume. Females treated with 300 mg/kg bw/day also showed a statistically significant increase in erythrocyte count. Females treated with 100 mg/kg bw/day also showed a statistically significant reduction in hemoglobin. The majority of individual values were within the historical control range and in the absence of any supporting microscopic findings to suggest a treatment-related effect in the spleen, the intergroup differences were considered to be of no toxicological importance.

Blood Chemistry
Animals of either sex treated with 300 mg/kg bw/day had increased alanine aminotransferase and aspartate animotransferase levels at the end of the treatment period. Males treated with 100 mg/kg bw/day also had increased alanine aminotransferase. With the exception of aspartate aminotransferase in the females, all intergroup differences achieved statistical significance and the majority of individual parameters exceeded the normal background ranges for rats of the strain and age used.

No such effects were detected in females treated with 100 mg/kg bw/day or animals of either sex treated with 10 mg/kg bw/day.

Males treated with 300 mg/kg bw/day also had a statistically significant reduction in bilirubin, however, all of the individual parameters were within the historical control range; therefore the intergroup differences were considered not to be toxicologically significant. All treated males had a statistically significant increase in chloride. Males treated with 300 mg/kg bw/day also had a statistically significant reduction in urea and males treated with 10 mg/kg bw/day had a statistically significant reduction in creatinine. All individual parameters were within normal background ranges and in the absence of a true dose related response in chloride and creatine or any histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Pathology
Necropsy
Neither the type, incidence nor distribution of macroscopic findings observed at necropsy indicated any effect of treatment at dosages up to 300 mg/kg bw/day.

One male treated with 300 mg/kg bw/day, three males and two females treated with 100 mg/kg bw/day, one control male and two control females had reddened lungs at necropsy. In the absence of a true dose related response and due to the presence of the finding in control animals, the intergroup differences were considered unrelated to treatment. Two females treated with 10 mg/kg bw/day had unilateral increased pelvic space of the kidney. In the absence of a similar effect at 300 mg/kg bw/day or any microscopic changes to suggest an effect of treatment in the kidney, these findings were considered not to be of toxicological importance.

Organ Weights
Animals of either sex treated with 300 mg/kg bw/day and males treated with 100 mg/kg bw/day showed an increase in liver weights both absolute and relative to terminal body weight with differences in males at 300 mg/kg bw/day achieving statistical significance. Although a true dose related response was not evident, the majority of individual body weight relative values at 300 mg/kg bw/day exceeded the background control ranges. In males at 100 mg/kg bw/day, individual weights exceeded background control ranges for absolute liver weights in three animals and in five animals for body weight relative liver weights.

No such effects were detected in animals of either sex treated with 10 mg/kg bw/day.

Females treated with 100 mg/kg bw/day showed a statistically significant reduction in liver weights, the majority of individual values were within normal background ranges and therefore were considered not to be of toxicological importance.


Histopathology
There were no treatment-related microscopic findings.

All of the histopathological findings encountered were considered to have arisen spontaneously or at post mortem.

Discussion
Administration of AS1100 by oral gavage at dose levels of 10, 100 and 300 mg/kg bw/day resulted in treatment related effects in animals of either sex at 300 mg/kg bw/day and in males at 100 mg/kg bw/day.

Males treated with 300 mg/kg bw/day had statistically significantly lower body weight gains on three occasions and overall body weight gains were reduced when compared to control animals. The magnitude of the difference from control, however, was not substantial enough to consider the effect adverse.

Animals of either sex treated with 300 mg/kg bw/day and males treated with 100 mg/kg bw/day had an increase in liver weights, both absolute and relative to terminal body weight, and an associated increase in alanine aminotransferase and aspartate aminotransferase. Individual values for both liver weights and hepatic enzymes exceeded background control ranges for the majority of animals, however, there were no microscopic abnormalities detected in the liver. The intergroup differences in liver weight and hepatic enzyme leves were, therefore, considered to represent an adaptive response to treatment and consequently were considered not to represent an adverse effect of treatment.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose tested
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: body weight; organ weights; clinical chemistry
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: body weight; organ weights; clinical chemistry
Critical effects observed:
not specified
Conclusions:
The oral administration of AS1100 to rats by gavage for a period of 90 consecutive days at dose levels of 10, 100 and 300 mg/kg bw/day resulted in treatment related effects in animals of either sex at 300 mg/kg bw/day and in males at 100 mg/kg bw/day. The No Observed Effect Level (NOEL) was considered to be 10 mg/kg bw/day for males and 100 mg/kg bw/day for females.

Lower body weight gains in males treated with 300 mg/kg bw/day were considered insufficient in magnitude to represent an adverse effect of treatment. The increased liver weights in animals of either sex treated with 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day, accompanied by associated increases in hepatic enzymes, in the absence of microscopic changes in the liver do not represent an adverse effect of treatment. The effects were considered to represent an adaptive response to treatment and therefore considered not to represent a serious risk to health.

For this reason, 300 mg/kg bw/day may be regarded as a 'No Observed Adverse Effect Level' (NOAEL) for animals of either sex.
Executive summary:

Introduction


The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:


i)  The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).


 


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 ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 10, 100 and 300 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).


 


The dose levels were chosen in collaboration with the Sponsor based on previous toxicity work including an OECD 422 study in which the No Observed Adverse Effect Level (NOAEL) was 300 mg/kg bw/day. Therefore, 300 mg/kg bw/day was selected for investigation as a high dose due to the prolonged exposure period.


 


Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals.


 


All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.


 


Results….


Mortality


There were no unscheduled deaths.


 


Clinical Observations


Neither the type, incidence or distribution of clinical signs apparent indicated an adverse effect of treatment at 10, 100 or 300 mg/kg bw/day.


 


Behavioral Assessment


There were no treatment-related changes in the behavioral parameters measured.


 


Functional Performance Tests


There were no toxicologically significant changes in functional performance.


 


Sensory Reactivity Assessments


There were no treatment-related changes in sensory reactivity.


 


Body Weight


There was no adverse effect of treatment on body weight development at 10, 100 or 300 mg/kg bw/day. 


 


Food Consumption


There was no obvious effect of treatment on food consumption at 10, 100 or 300 mg/kg bw/day.


 


Water Consumption


Daily visual inspection of water bottles did not reveal any overt differences in water consumption at 10, 100 or 300 mg/kg bw/day.


 


Ophthalmoscopy


There were no treatment-related ocular changes.


 


Hematology


There were no toxicologically significant effects detected in the hematological parameters examined.


 


Blood Chemistry


Animals of either sex treated with 300 mg/kg bw/day and males treated with 100 mg/kg bw/day had increased alanine aminotransferase and aspartate animotransferase at the end of the treatment period. No such effects were detected in females treated with 100 mg/kg bw/day or animals of either sex treated with 10 mg/kg bw/day.


 


Necropsy


Neither the type, incidence nor distribution of macroscopic findings observed at necropsy indicated any effect of treatment at dosages up to 300 mg/kg bw/day.


 


Organ Weights


Animals of either sex treated with 300 mg/kg bw/day showed an increase in liver weights both absolute and relative to terminal body weight with differences in males achieving statistical significance. Males treated with 100 mg/kg bw/day also showed an increase in both absolute and body weight relative liver weights. No toxicologically significant effects were detected in females treated with 100 mg/kg bw/day or animals of either sex treated with 10 mg/kg bw/day.


 


Histopathology


There were no treatment related microscopic abnormalities detected.


All of the histopathological findings encountered were considered to have arisen spontaneously or at post mortem.


 


Conclusion


The oral administration of AS1100 to rats by gavage for a period of 90 consecutive days at dose levels of 10, 100 and 300 mg/kg bw/day resulted in treatment related effects in animals of either sex at 300 mg/kg bw/day and in males at 100 mg/kg bw/day. The No Observed Effect Level (NOEL) was considered to be 10 mg/kg bw/day for males and 100 mg/kg bw/day for females.


 


Lower body weight gains in males treated with 300 mg/kg bw/day were considered insufficient in magnitude to represent an adverse effect of treatment. The increased liver weights in animals of either sex treated with 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day, accompanied by associated increases in hepatic enzymes, in the absence of microscopic changes in the liver do not represent an adverse effect of treatment. The effects were considered to represent an adaptive response to treatment and therefore considered not to represent a serious risk to health.


For this reason, 300 mg/kg bw/day may be regarded as a 'No Observed Adverse Effect Level' (NOAEL) for animals of either sex.

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: U.S. EPA, OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (females) and Charles River, UK (males)
- Age at study initiation:10-12 weeks (males/females)
- Weight at study initiation: m (314.4-316.7); f (195.8-199.5)
- Fasting period before study: no data
- Housing:individually in type M III polycarbonate cages supplied by Becker & Co., Castrop Rauxel, Germany (floor area of about 800 cm2)
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 6-8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24 °C
- Humidity (%):30-70%
- Air changes (per hr):10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare the solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (propylene glycol) was filled up to the desired volume, subsequently mixed using a magnetic stirrer.
The test-substance solutions were prepared in such intervals that the stability was guaranteed.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle:

group dose conc. Male Female Male
0 0 0 5 10 10
1 100 2.0 5 10 10
2 300 6.0 5 10 10
3 1000 20.0 5 10 10

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the test-substance preparations
The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE as a part of the study.
The stability of the test substance in propylene glycol stored deep frozen over a period of 7 days followed by 4 hours at room temperature was demonstrated before the start of the study (Project No.: 01Y0471/09Y004).
Concentration control and homogeneity analyses of the test-substance preparations were performed in samples of all concentrations at the start and at the end of the administration period.

Food analysis
The food used in the study was assayed for chemical and microbiological contaminants.

Drinking water analysis
The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for bacteria by a contract laboratory. The German “Trinkwasserverordnung” (Drinking Water Regulation) will serve as the guideline for maximum tolerable contaminants.

Bedding and enrichment analysis
The bedding and the enrichment are regularly assayed for specific contaminants (chlorinated hydrocarbons and heavy metals).
Duration of treatment / exposure:
females were treated for 49 days and males for 35
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10 m / 10 f
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: In a previously performed 14-days test study (Project No.: 10C0471/09069) ASA was administered by gavage to groups of 3 male and 3 female Wistar rats at dose levels of 0, 100, 300, 1000 mg/kg body weight/day (mg/kg bw/d). Propylene glycol swas the vehicle.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before and after treatment for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:before and after treatment. The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams.The day of littering was considered to be a 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

BODY WEIGHT: Yes
- Time schedule for examinations:once a week. The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

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
Once a week (in a period of 7 days) for male and female parental animals, with the following exceptions:
•Food consumption was not determined during the mating period (male and female F0 animals).
•Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
•Food consumption of F0 females, which gave birth to a litter, was determined on PND 0 and 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:towards the end of the administration period (study days 35 (males) and 49 (females))
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood:
- Animals fasted: Yes / No / No data
- How many animals: 5 randomly selected
- Parameters c Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:towards the end of the administration period (study days 35 (males) and 49 (females))
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume were examined.

NEUROBEHAVIOURAL EXAMINATION:No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes


Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland
11. Prostate
12. Seminal vesicles with coagulation glands
13. Spleen
14. Testes
15. Thymus
16. Thyroid glands (with parathyroid glands)
17. Uterus

Organ/tissue fixation
The following organs or tissues of parental animals were fixed in 4% formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulation glands
9. Colon
10. Duodenum
11. Epididymides (modified Davidson`s solution)
12. Esophagus
13. Eyes with optic nerve
14. Female mammary gland
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (mesenteric and axillary lymph nodes)
24. Nose (nasal cavity)
25. Ovaries
26. Oviducts
27. Pancreas
28. Parathyroid glands
29. Pharynx
30. Pituitary gland
31. Prostate
32. Rectum
33. Salivary glands (mandibular and sublingual glands)
34. Seminal vesicle
35. Sciatic nerve
36. Skeletal muscle
37. Spinal cord (cervical, thoracic and lumbar cords)
38. Spleen
39. Sternum with marrow
40. Stomach (forestomach and glandular stomach)
41. Testes (modified Davidson’s solution)
42. Thymus
43. Thyroid glands
44. Trachea
45. Urinary bladder
46. Uterus
47. Vagina


HISTOPATHOLOGY: Yes
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulation gland
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Peyer’s patches
20. Prostate
21. Rectum
22. Sciatic nerve
23. Seminal vesicle
24. Spleen
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Stomach (forestomach and glandular stomach)
27. Testes
28. Thymus
29. Thyroid glands (with parathyroid glands)
30. Trachea
31. Urinary bladder
32. Uterus
33. Vagina

Other examinations:
CLINICAL PATHOLOGY
In the morning, blood was taken from the retroorbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results.
The results of the clinical pathology examinations are expressed in units of the International System (SI).
The examinations were carried out in 5 animals per test group and sex.
Statistics:
Statistics of the clinical examinations
DUNNETT-test , FISHER'S EXACT test, WILCOXON-test, KRUSKAL-WALLIS test

Statistics of clinical pathology
FISHER'S EXACT test, KRUSKAL-WALLIS test

Statistics of pathology
KRUSKAL-WALLIS test, WILCOXON-test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No mortality, piloerection, poor general state in female animals during lactation, insufficient maternal care
Mortality:
mortality observed, treatment-related
Description (incidence):
No mortality, piloerection, poor general state in female animals during lactation, insufficient maternal care
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Increased platelet counts in rats of both sexes, increased WBC counts, absolute neutrophil, relative and absolute monocyte as well as relative and absolute LUC counts in males
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Reduced prothrombin time in males / Decreased total protein and albumin values in males / Increased cholesterol values in males / Increased ALT activities and bilirubin values in rats of both sexes / Increased SGGT activities in males
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalyses parameters were measured
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Diffuse squamous cell hyperplasia in the forestomach of all males and females. Multifocal atypical bile duct hyperplasia in the liver of 8 males and of all females.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No animal died prematurely during the study period.In test group 3 (1000 mg/kg bw/d), salivation after treatment was observed in 6 males from study week 1 onwards and in 2 males in study weeks 3 and 4. Three male animals in test group 2 (300 mg/kg bw/d) showed salivation after treatment in study weeks 3 and 4. In females no salivation was observed during the premating period. One male of test group 0 (control) showed an eye injury in study week 4.
Salivation after treatment was observed in 3 to 4 female animals of test group 3 (1000 mg/kg bw/d) from GD 3 onwards. Salivation after treatment was also observed in 1 female animal of test group 2 (300 mg/kg bw/d) from GD 8 onwards.
Five female animals of test group 3 (1000 mg/kg bw/d), i.e. animal Nos. 132, 134, 137, 138 and 139, showed insufficient maternal care of pups. In two litters no more pups were alive on PND 1 (animal No. 139) and 2 (animal No. 138). Four of these animals also showed piloerection, i.e. animal Nos. 132, 134, 138 and 139. In addition urine smeared anogenital region was observed in female No. 139. Three dams of test group 3 (1000 mg/kg bw/d), i.e. animal Nos. 135, 136 and 139, showed salivation after treatment. Animal No. 124 of test group 2 (300 mg/kg bw/d) showed insufficient maternal care, piloerection and salivation after treatment. Poor general state, piloerection and insufficient maternal care of pups were observed in animal No. 111 of test group 1 (100 mg/kg bw/d).

BODY WEIGHT AND WEIGHT GAIN
No significant changes in body weight and body weight change values for male and female animals were observed during the entire study period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No significant changes in food consumption values for male and female animals were observed during the entire study period. Although not significantly decreased, food consumption in female animals of test group 3 (1000 mg/kg bw/d) was only 78% compared to test group 0 (control) during lactation.

FOOD EFFICIENCY
no data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
no data

OPHTHALMOSCOPIC EXAMINATION
no data

HAEMATOLOGY
In male rats of test group 3 (1000 mg/kg bw/d) total white blood (WBC) counts were increased which was mainly due to an increase of the absolute neutrophil counts. In addition, absolute and relative monocyte counts (relative counts not significant) as well as absolute and relative large unstained cell (LUC) counts were higher in males of test group 3 (1000 mg/kg bw/d) compared to controls. In females of test group 2 (300 mg/kg bw/d) relative monocyte counts were lower compared to controls, but this decrease was not dose-dependent, and it was not accompanied by any other alteration in the differential blood cell counts in these animals. Therefore, this change was regarded as incidental and not treatment-related.
In male and female rats of test group 3 (1000 mg/kg bw/d) platelet counts were increased (not significantly in females).
In males of test group 3 (1000 mg/kg bw/d) the prothrombin time (Hepato Quick’s test) was reduced compared to controls.

CLINICAL CHEMISTRY
In male and female rats of test groups 2 and 3 (300 and 1000 mg/kg bw/d) the alanine aminotransferase (ALT) activity was increased. The ALT values in rats of test group 2 (300 mg/kg bw/d) were within (females) or at the border of (males) the historical control range (ALT males: 0.46-0.94 µkat/L; ALT females: 0.42-0.73 µkat/L, PART III, Supplement). In males of this test group marginal higher ALT activities were the only altered parameter. In addition to the ALT activity increase in females of test group 2 (300 mg/kg bw/d), significantly higher total bilirubin values were measured, but these values were also in the historical control range (bilirubin: 2.30-3.46 µmol/L, PART III, Supplement). Therefore, the ALT activity increases in rats of both sexes of test group 2 (300 mg/kg bw/d) as well as the higher bilirubin values in females of the same test group were regarded as incidental and not treatment-related (ECETOC Technical Report No. 85, 2002). In 2 males of test group 3 (1000 mg/kg bw/d; Nos. 32 and 35) the -glutamyltransferase (SGGT) activity was increased.
In males of test group 3 (1000 mg/kg bw/d) albumin and total protein values (not significant) were decreased, whereas cholesterol values in males of the same test group were increased.
The bilirubin values in females of test group 3 (1000 mg/kg bw/d) were higher compared to controls and at least 1 male rat (No. 35) in the same test group did also have a high total bilirubin level.
In females of test groups 1 and 2 (100 and 300 mg/kg bw/d) lower triglyceride levels were measured compared to controls, but this decrease was not dose-dependent and, therefore, it was regarded as incidental rather than treatment-related.

URINALYSIS
No treatment-related changes among urinalyses parameters were measured.

NEUROBEHAVIOUR
no data

ORGAN WEIGHTS
All mean absolute weight parameters in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) did not show significant differences when compared to test group 0 (control).
When compared to test group 0 (control; set to 100%), the mean relative weights of following organs were significantly increased: kidney, testes (male); liver (female)
All other mean relative weight parameters did not show significant differences when compared to test group 0 (control).

The increase of the relative kidney weight in males of test group 1 (100 mg/kg bw/d) was regarded to be incidental as no dose-response relationship occurred. The increased relative liver weight in females of test group 2 (300 mg/kg bw/d) was also considered to be incidental because the mean absolute weight was not significantly increased and no histopathological correlate was present.
For the increase of the relative testes weight in males of test group 3 (1000 mg/kg bw/d) there were no histopathological correlates. Furthermore, the absolute weight of testes did not show significant differences. Therefore, the increased testes weight was related to the slightly but not significantly decreased terminal body weight (-5%) in these males.

GROSS PATHOLOGY
Forestomach: Three males of test group 2 (300 mg/kg bw/d) and 6 males of test group 3 (1000 mg/kg bw/d) showed a yellow-white deposition on the forestomach epithelium. A thickening of the forestomach wall was observed in all males of test group 3 (1000 mg/kg bw/d).
Duodenum: The duodenal wall was slightly thickened in 7 males of test group 3 (1000 mg/kg bw/d).
All other gross lesions occurred singly and were considered to be incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
Forestomach: A diffuse squamous cell hyperplasia was observed in the forestomach of 1 control female and of 1 female in test group 1 (100 mg/kg bw/d). In test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d), incidence and severity of diffuse squamous cell hyperplasia were increased dose-related. The squamous cell hyperplasia was characterized by an increased number of epithelial cells showing small exophytic structures into the lumen or small endophytic finger-like projections. The hyperplasia was accompanied by an infiltration of some lymphoid cells and granulocytes. The hyperplasia correlated with the thickening of the forestomach wall that was grossly diagnosed in all males of test group 3 (1000 mg/kg bw/d). The increased occurrence of squamous cell hyperplasia was considered to be treatment-related.
The squamous cell hyperplasia occurred mostly in combination with hyperkeratosis. The macroscopically diagnosed yellow-white deposition on the forestomach epithelium corresponded histopathologically with a diffuse orthokeratotic hyperkeratosis, characterized by an increased thickening of the superficial non-nucleated keratin layer. The incidence of hyperkeratosis was increased treatment-related in males of test group 2 (300 mg/kg bw/d) as well as in males and females of test group 3 (1000 mg/kg bw/d).
The occurrence of the minimal squamous cell hyperplasia in the forestomach of 1 control female and 1 female of test group 1 (100 mg/kg bw/d) as well as the occurrence of hyperkeratosis in 1 control female, in 2 females of test group 1 (100 mg/kg bw/d) and in 2 females in test group 2 (300 mg/kg bw/d) were considered to be incidental.

Liver: A multifocal atypical bile duct hyperplasia was observed in 2 males of test group 2 (300 mg/kg bw/d) as well as in 8 males and all females of test group 3 (1000 mg/kg bw/d). This finding was characterized by a multifocal proliferation of bile ducts in the portal region. The bile duct epithelium seemed slightly atypical. It was single layered, often cuboidal and slightly basophilic. Some mitotic figures and necrotic cells were present within the epithelium. The hyperplasia was associated with periductular fibrosis and periductular lymphohistiocytic infiltrates.
Furthermore, a minimal centrilobular, hepatocellular hypertrophy was observed in 8 males and 7 females of test group 3 (1000 mg/kg bw/d).

The occurrence of multifocal atypical bile duct hyperplasia and of centrilobular, hepatocellular hypertrophy was considered to be treatment-related.

Thyroid glands: A minimal or slight follicular hypertrophy/hyperplasia was observed in 1 control male, in 2 males of test group 1 (100 mg/kg bw/d), in one male of test group 2 (300 mg/kg bw/d), and in 4 males of test group 3 (1000 mg/kg bw/d). In affected animals the number of small follicles was slightly increased or the follicular epithelium was higher, varying in size from cuboidal cells to columnar cells.
For the slightly increased incidence of hypertrophy/hyperplasia in males of test group 3 (1000 mg/kg bw/d), a treatment related effect could not be ruled out.

Duodenum: For the macroscopically described thickening of duodenal wall in 7 males of test group 3 (1000 mg/kg bw/d), there was no histopathological correlate.

All other findings occurred either individually or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: haematology; gross pathology; histopathology;
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on fertility observed
Dose descriptor:
dose level:
Effect level:
>= 300 - <= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See remarks
Remarks on result:
other: Diffuse squamous cell hyperplasia in the forestomach of males and females and multifocal atypical bile duct hyperplasia in the liver of males
Dose descriptor:
dose level:
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See remarks
Remarks on result:
other: See remarks in basis for effect level
Remarks:
Increased platelet counts in rats of both sexes Increased ALT activities and bilirubin values in rats of both sexes Increased SGGT activities in males Increased WBC counts, absolute neutrophil, relative and absolute monocyte as well as relative and absolute LUC counts in males Reduced prothrombin time in males Decreased total protein and albumin values in males Increased cholesterol values in males
Critical effects observed:
not specified

Functional observational battery

Home cage observations:

No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

  

Open field observations:

One female animal of test group 2 (300 mg/kg bw/d;animal No. 128) showed piloerection. All other male and female animals of all test groups did not reveal any test substance-related findings during home cage observation.

 

Sensorimotor tests/reflexes:

There were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore,these observations were considered as being incidental.

 

Quantitative Parameters:

No test substance-related impaired parameters were observed in male and female animals of all test group

 Motor activity measurement

There were no significant deviations concerning the overall motor activity (summation of all intervals) in the male and female animals of all test groups in comparison to the concurrent control group.

Regarding single intervals, in males of test group 1 (100 mg/kg bw/d) one isolated significantly increased value was measured at interval 10. This finding was considered as being incidental since the overall motor activity was not changed and no findings were observed for female animals.

Conclusions:
The results of this OECD 422 study are, that the no observed adverse effect level (NOAEL) for systemic toxicity in F0 males was 100 mg/kg bw/d due to pathological findings at 300 mg/kg bw/d and above including increased blood values and squamous cell hyperplasia in the forestomach of all animals. However, the NOAEL for systemic toxicity in F0 females could not be established, although the dose of 100 mg/kg bw/d was without pathological findings. This was due to the noted insufficent maternal care at all dose levels correlates with increased pup mortality. The cause for the insufficent maternal care is presently undecided as the effect may be due to systemic maternal toxicity or due to an effect on the pups via lactation. The NOAEL for fertility in male and female Wistar rats was 1000 mg/kg bw/d.
Executive summary:

In a study performed according to OECD 422 and GLP, the test substance was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 parental animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; vehicle control; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d in propylene glycol, respectively. As results, the no observed adverse effect level (NOAEL) for systemic toxicity in F0 males was 100 mg/kg bw/d due to pathological findings at 300 mg/kg bw/d and above including increased blood values and squamous cell hyperplasia in the forestomach of all animals. However, the NOAEL for systemic toxicity in F0 females could not be established, although the dose of 100 mg/kg bw/d was without pathological findings. This was due to the noted insufficent maternal care at all dose levels correlates with increased pup mortality. The cause for the insufficent maternal care is presently undecided as the effect may be due to systemic maternal toxicity or due to an effect on the pups via lactation. The NOAEL for fertility in male and female Wistar rats was 1000 mg/kg bw/d.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
studies performed according to GLP
System:
gastrointestinal tract
Organ:
stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The overall NOAEL as selected is based on the EOGRTS study as summarized under reproduction toxicity and in the text below.


In a 90-day study ASA was administered by gavage to 10 rats/sex/group at dose levels of 10, 100 and 300 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).


Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals.


All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.


There were no effects on mortality, clinical signs, behaviour (functional and sensory tests), body weight (gain), food- and water consumption, haematology, macroscopic and histopathological examinations.


At the end of treatment increased liver weights in animals of either sex treated with 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day, accompanied by associated increases in hepatic enzymes (ALAT and ASAT) were seen. In the absence of microscopic changes in the liver this does not represent an adverse effect of treatment. The effects are considered to represent an adaptive response to treatment. For this reason, the NOAEL is set at 300 mg/kg bw (Harlan 2014)


 


In a study performed according to OECD 422, the test substance was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 parental animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; vehicle control; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d in propylene glycol, respectively. As results, the no observed adverse effect level (NOAEL) for systemic toxicity in F0 males was 100 mg/kg bw/d due to pathological findings at 300 mg/kg bw/d and above including increased blood values and squamous cell hyperplasia in the forestomach of all animals. However, the NOAEL for systemic toxicity in F0 females could not be established, although the dose of 100 mg/kg bw/d was without pathological findings. This was due to the noted insufficient maternal care at all dose levels correlates with increased pup mortality. The cause for the insufficient maternal care is presently undecided as the effect may be due to systemic maternal toxicity or due to an effect on the pups via lactation. The NOAEL for fertility in male and female Wistar rats was 1000 mg/kg bw/d (BASF 2010).


A cross fostering study with a study design similar to OECD 422 was to assess the influence of the substance when administered by oral gavage at dose levels of 100, 300 or 1000 mg/kg/day on reproductive performance in the Han Wistar rat to assist in dose level selection for a main extended one generation reproductive toxicity study (OECD443) and to investigate the observations of insufficient maternal care and increased offspring mortality seen in a previously performed OECD 422 study (BASF 2010).The general condition and macro-pathology of F0 parental animal and F1 offspring were unaffected by administration of ASA.  F0 parental animals at 1000 mg/kg/day showed low body weight gain and food consumption during the period before pairing, this continued for females during the second half of gestation.  On Day 1 of lactation females at 1000 mg/kg/day had low mean body weight but subsequent gain up to weaning was unaffected by treatment.
There were no adverse effects on mating performance, fertility, pre-coital interval, gestation length or gestation index. However, on Day 1 of lactation females receiving 1000 mg/kg/day had a low number of implantations and total litter size when compared with Controls (2 outliers) and the mean body weight for both male and female offspring derived from females receiving 1000 mg/kg/day was significantly low despite the lower litter size.
In order to try and elucidate the maternal role in the mortality of offspring seen in a previous OECD 422 study (BASF 2010) treated litters were fostered to Control females and vice versa; in addition, a sub-group of females were sham fostered remaining with the original dam in order to ascertain any effects following litter disturbance.
Following fostering procedures there was no significant difference for within-litter offspring mortality. The incidence of scattered offspring was slightly higher for litters with F0 females receiving 1000 mg/kg/day in both the sham and cross-fostered sub-groups. There was no effect on litter size, offspring survival or sex ratio. Absolute body weights and body weight gain for offspring at 1000 mg/kg/day was significantly low for litters when compared with their respective Controls i.e sham or cross-fostered.  Control litters fostered to females receiving 300 mg/kg/day also showed low weight gain.
In view of these findings for either sham fostered high dose litters or Control litters with high dose females it was concluded that the effect on the offspring was principally due to the maternal dose effect (Covance 2021).


In a study according to OECD 443 the F0 generation, 25 rats/sex/group received ASA at dose levels of 100, 300 or 1000 mg/kg bw. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. A similarly constituted Control group received the vehicle, propylene glycol, at the same volume dose as treated groups.
The F1 generation comprised of two cohorts:


Cohort 1A: 20 male and 20 female progeny were selected from each dose group and continued to receive ASA at dose levels of 0 (Control), 100, 300 or 1000 mg/kg bw from weaning until scheduled sacrifice at approximately Week 13 of age. A similarly constituted Control group received the vehicle, propylene glycol, at the same volume dose as treated groups.
Cohort 1B: 20 male and 20 female progeny were selected from each dose group and continued to receive ASA at dose levels of 0 (Control), 100, 300 or 1000 mg/kg bw from weaning until scheduled sacrifice at approximately Week 14 of age. A similarly constituted Control group received the vehicle, propylene glycol, at the same volume dose as treated groups.


F0 generation


In the F0 pale faeces was observed in both sexes at different times during treatment. Body weight (gain) was slightly low in females at 300 and 1000 mg/kg bw during gestation. Minimal effects on food consumption were noted occasionally in females at 300 and 1000 mg/kg bw during gestation and lactation.
At scheduled termination males at 300 or 1000 mg/kg/day showed low mean haematocrit and erythrocyte counts, with high mean cell haemoglobin (concentration). Males at 1000 mg/kg/day also had a high mean platelet count and high total white blood cell counts. In males and females at 1000 mg/kg bw ALP, ALAT and bile acid levels were high and A/G ratio was slightly low. At 300 mg/kg bw both sexes also showed high ALAT levels.  Males at 1000 mg/kg bw had high ASAT levels.
Mean serum Thyroid stimulating hormone and thyroxine levels showed no adverse effects of treatment.
At 1000 mg/kg bw cauda epididymal sperm numbers were statistically significantly low with high testes spermatid numbers This could indicate a potential delay in progression of spermatids from the testis to the epididymis, however this had no adverse effects on sperm motility/motion or morphology.
There were no treatment related effects on organ weights. Macroscopic changes in the stomach of both sexes treated with 100, 300 or 1000 mg/kg bw comprised of depression and/or thickened stomach. In the duodenum an abnormal color was noted  in both sexes at 300 mg/kg bw and in males treated with 1000 mg/kg/day. The livers of males administered 300 or 1000 mg/kg/day had dark/pale areas. Microscopic examination of the non-glandular stomach showed epithelial hyperplasia (minimal to marked) was observed in both sexes at 100, 300 or 1000 mg/kg bw accompanied by hyperkeratosis, occasional erosions, ulceration, a single case of papilloma and by inflammation in females administered 300 or 1000 mg/kg bw. In the liver of both sexes centrilobular hypertrophy was observed at 100, 300 or 1000 mg/kg bw. Bile duct hyperplasia was observed in both sexes administered 1000 mg/kg bw, in males associated with fibrosis and/or granulocytic inflammatory infiltrate surrounding the bile ducts and/or periportal areas with occasional cases of pigment in the bile duct. Hepatocellular necrosis and vacuolisation was also noted in males at 1000 mg/kg bw  Liver changes observed in the males were of greater severity. Minimal to slight villous hypertrophy was observed in the duodenum of both sexes at 100, 300 or 1000 mg/kg bw and 4 control females.  
For the F0 generation there were no treatment related effects on reproductive performance (sperm motility, morphology and testes sperm counts, estrous cycle, pre-coital interval, mating performance, fertility, gestation length and gestation index).
Based on these findings the NOAEL for generic toxicity is set at 100 mg/kg bw, while the NOAEL for reproduction is 1000 mg/kg bw


Litter Responses


The general condition of offspring, litter size, offspring survival, sex ratio and ano-gentital distance were unaffected by treatment. On Day 1 of age the mean body weight for male offspring at 1000 mg/kg bw was slightly but statistically significantly low (<10%) compared to controls; this difference was not apparent for female offspring.  Subsequent body weight gain up to Day 4 of age was similar across the groups, however body weight gain at 1000 mg/kg bw for male offspring from Day 4 to 21 of age was low (ca 8% lower than controls). There were no organ weight differences on Day 22 of age and no macroscopic findings that could be related to parental treatment with ASA.
Mean serum thyroid stimulating hormone and thyroxine levels showed no adverse effects of treatment.
Based on these findings the NOAEL for developmental toxicity is set at 1000 mg/kg bw


F1 Generation


There were no treatment related effects on mortality, clinical signs, sexual maturation, estrous cycle and sperm parameters
At weaning on Day 21 until Day 28 (day 1 of the formal start of the F1) body weights for selected F1 males at 1000 mg/kg bw and F1 females at 300 or 1000 mg/kg bw were low, but effects on body weight gain during the exposure period were considered negligible. Males at 100, 300 or 1000 mg/kg bw showed low mean haematocrit. In addition males at 300 and 1000 mg/kg bw showed high neutrophil counts and short prothrombin times. Males at 1000 mg/kg bw had a high mean platelet count. Females at all dose levels had a low prothrombin time and at 1000 mg/kg bw had a low haematocrit and low haemoglobin.
ALAT levels were high in both sexes at 300 and 1000 mg/kg bw and albumin and protein levels were low at 1000 mg/kg bw. In males at 1000 mg/kg bw high bile acid levels, low urea and low phosphorous levels were reported. Females in all treated groups had slightly high potassium levels.
Mean serum Thyroid stimulating hormone and thyroxine levels showed no adverse effects of treatment.
At 1000 mg/kg/day, urinary pH was low in males and females and sodium levels were slightly low in males.
At scheduled termination Cohort 1A females at 300 or 1000 mg/kg bw showed slightly high body weight relative adrenal weights. Substance-related macroscopic changes were noted in the stomach and comprised of depression (both sexes treated with 1000 mg/kg bw) and thickened stomach (both sexes at 300 or 1000 mg/kg bw).
There were no differences observed in the spleenimmunophenotyping parameters across the F1 Cohort 1A  treatment groups that could be related to treatment.Microscopic findings in the nonglandular stomach consisted of epithelial hyperplasia in both sexes at 100, 300 or 1000 mg/kg bw by hyperkeratosis and occasional edema in both sexes, a single case of erosion in males administered 300 mg/kg/day, and ulceration in males administered 1000 mg/kg/day.
In the liver of both sexes centrilobular hypertrophy was observed at 100, 300 or 1000 mg/kg bw. Bile duct hyperplasia and the fibrosis associated with granulocytic inflammatory infiltrate surrounding the bile ducts were observed in the liver of both sexes administered 1000 mg/kg bw with males displaying a higher incidence. Hepatocellular necrosis (minimal to slight) was also noted in one female administered 100 or 300 mg/kg bw and in both sexes administered 1000 mg/kg bw. Minimal to slight (one male) villous hypertrophy was observed in the duodenum of both sexes administered 100, 300 or 1000 mg/kg bw. 


Based on these findings the NOAEL for generic toxicity was set at 100 mg/kg bw (Covance 2021).


 

Justification for classification or non-classification

There are conclusive and sufficient data for classification of with regard to repeated dose toxicity.


The substance is not classified for this endpoint in accordance with the CLP Regulation (EC) No 1272/2008.