Dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium

Endpoint summary

Description of key information

A 90 -day oral toxicity study is available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 29th 2012 to September 9th 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

-Lot: 1314
-Purity: 30.8% (w/w)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CHARLES RIVER LABORATORIES JAPAN, INC., Atsugi.
- Age at study initiation: 5 weeks
- Housing: one male/female in one stainless cage (260 W x 380 D x 180 H(mm))
- Diet: Ground diet
- Water: Sterile water (treated by 1 μm filter and UV)
- Acclimation period: 6 days for male, 7 days for female

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3ºC (Actual temperature: 21.5-24.6ºC)
- Humidity: 55 ± 10% (Actual humidit: 52-64%)
- Air changes: ≥ 10 per hour
- Photoperiod: 12 hours dark / 12 hours light (From 7 am to 7 pm)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): twice November 5th, 2012 and December 25th, 2012)
- Mixing appropriate amounts with (Type of food): Diets were prepared for each concentration group. The test substance was mixed with the diet using a middle size mixer to obtain a pre-mix. Then the pre-mix was diluted by the diet using a large size mixer to obtain testing diets in each concentration.
- Storage temperature of food: 2-6ºC
- Storage period: Analytical results showed that the test substance was stable at least for 53 days in the diet.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in each diet was determined by HPLC and it was concluded that each test concentration was correctly prepared.

Duration of treatment / exposure:

90 days

Frequency of treatment:

In diet

Dose / conc.:

0 other: %

Remarks:

Untreated controls

Dose / conc.:

0.056 other: %

Remarks:

Actual dose recieved: 41 mg/kg bw/d (male), 46 mg/kg bw/d (female)

Dose / conc.:

0.167 other: %

Remarks:

Actual dose recieved: 120 mg/kg bw/d (male), 150 mg/kg bw/d (female)

Dose / conc.:

0.5 other: %

Remarks:

Actual dose recieved: 380 mg/kg/day (male), 440 mg/kg/day (female)

No. of animals per sex per dose:

The number of animals in each group was 10 males and 10 females. In addition, a satellite group (recovery group) of 5 male and 5 female animals was established for the control group and the highest dose group in order to evaluate the reversibility, persistence and delayed occurrence of the toxic effects.

Control animals:

yes, plain diet

Details on study design:

The treatment period was planned as 90 days (13 weeks), and the start day of treatment and the start week of treatment were planned as Day 0 of treatment and Week 1 of treatment, respectively. The recovery period was planned as 14 days after completion of the last treatment, and the day following the last treatment and the start week of recovery were planned as Day 1 of recovery and Week 1 of recovery, respectively.

Animal assignment was done on the start day of treatment. During the acclimation period, animals were quarantined based on the clinical observations and the body weight measurement, and the requisite number (50 males and 50 females) of animals with body weight close to the mean of all animals were selected from healthy, well grown animals in good physical conditions. Then, animals were stratified by body weight and randomly assigned to each group so that body weights might be comparable among the groups. The mean body weight (a range of body weights) at the start of treatment was 148 (138 to 156) g for males and 139 (129 to 150) g for females.

Positive control:

None

Observations and examinations performed and frequency:

Case side observation:
- Examined for any toxicity, ill-health, death or behavioural changes
- Time schedule: Daily, Twice (morning and evening)

Functional observational battery:
(1) Close clinical observations
On the start day of treatment and then, once a week during the treatment period and the recovery period, all animals were subjected to close clinical observations. That is, in addition to cage side observations, ease of removal from a cage, vocalization, physical tension (relaxation to tetanus), skin (color), coat, fur soiled with urine in the lower abdominal region, fur soiled with feces around the anal area, palpebral closure, exophthalmos, eye discharge, oral and nasal discharge (soiled fur), lacrimation, salivation, piloerection, breathing, gait (staggering), posture, exploratory behavior (awareness), tremor, convulsion, abnormal behavior (self-biting, walking backwards etc.), stereotyped behavior (excessive grooming, repeated circling etc.), alertness, locomotor activity (activity), clouding of consciousness (stupor, catalepsy, coma), muscle tone of four limbs, urination(frequency) and defecation (frequency), a total of 29 items, were observed while removing from the cage and in the standard observation field outside the cage [Aluminum open field (370 W×560 D×40 Hmm)].The changes were evaluated and recorded based on the scores (Appendix 7).
Animals were randomly assigned to observation numbers, and the cage cards showing the group and animal No. were replaced with those showing only the observation number by a person other than the observer so that the observer might be able to observe animals in numerical order of the observation numbers in a blinded manner.

(2) Sensorimotor function observations
At Week 13 of treatment in animals to be tested at the end of the treatment period and at week 2 of recovery in animals to be tested at the end of the recovery period, visual response (approaching response to a stick getting close to the face), auditory response (startle reaction to a light tapping on the cage with tweezers), touch response (response to a touch on the lower back region of animals), pain response (escape or vocalization reactions when pinched the tail base with tweezers), pupillary reflex (pupillary reaction to light) and righting reflex (a reflex that corrects the orientation of the body from the supine position on the plane surface) were evaluated in a blinded manner, and the reactions were scored (Appendix 8) and recorded.

(3) Grip strength and locomotor activity
At Week 13 of treatment in animals to be tested at the end of the treatment period and at Week 2 of recovery in animals to be tested at the end of the recovery period, forelimb and hindlimb grip strength (Grip strength meter for rats and mice, MK-380R/FR, Muromachi Kikai Co., Ltd.) and locomotor activity (Activity Monitoring System, SUPERMEX, Muromachi Kikai Co., Ltd) were measured.
Grip strength was determined for the forelimbs and hindlimbs three times each in a blinded manner and the mean of three measurements was obtained as a value of the relevant animal. Furthermore, locomotor activity was measured by detecting the movement of animals by means of a far-infrared ray sensor at an interval of 10 minutes, and the frequency of locomotion for 60 minutes was calculated.
 
BODY WEIGHT:
- Time schedule for examinations: On day 0 and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal for 48 hours was determined weekly from the two days before the body weight examination and mean daily diet consumption calculated.
- Compound intake calculated as mean values from the concentration of test substance, the consumption and body weight gain as in mg/kg/day

OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: week 13. As for the recovery, week 2
- Dose groups that were examined: Control, highest dose (No abnormalities were observed)
- Examinations included observation of the anterior chamber, optic media and ocular fundus.

URINALYSIS
- Time schedule for collection of urine: week 12. As for the recovery, week 2
- Metabolism cages used for collection of urine: Yes for 3 hours and 18 hours.
- Parameters examined: For the 3-hour urain, appearance, pH, protein, blood, glcose, ketones, bilirubin and urobilinogen
For the 18-hour urain, volume, specific gravity, sodium and potassium

Hematology
Blood was drawn from the abdominal aorta of animals under isoflurane inhalation anesthesia on the day following the end of treatment period and the day following the end of recovery period. Animals were fasted from 5 p.m. on the day before blood sampling, except water was given ad libitum. Blood collected from animals was divided into 3 aliquots. An aliquot of the blood was anticoagulated with EDTA-2K, and red blood cell counts (a method based on the detection of changes in electric resistance), hemoglobin content (sodium lauryl sulfate-hemoglobin method), hematocrit value (a method detecting peaks of pulses indicative of red blood cells), mean corpuscular volume (MCV, calculated value), mean corpuscular hemoglobin (MCH, calculated value), mean corpuscular hemoglobin concentration (MCHC, calculated value), platelet counts (a method based on the detection of changes in electric resistance), white blood cell counts, reticulocyte counts and differential leukocyte counts (flow cytometry for these cell counting) were measured using a multi-parameter automated hematology analyzer (XT-2000iV, Sysmex Corporation). In addition, an aliquot of the blood was anticoagulated with 3.8% sodium citrate, centrifuged, and plasma was obtained. Then, prothrombin time (PT, Quick one-stage method) and activated partial thromboplastin time (APTT, using ellagic acid for activation) were measured by using an automated coagulation analyzer (ST art-4, Roche Diagnostics GmbH).

Blood biochemistry
Serum was obtained from the remaining aliquot of the blood by centrifugation. Then, total protein (the biuret method), albumin (BCG1) method), A/G ratio (calculated value), glucose [the enzyme method (GluK2)-G-6-PDH3) method)], total cholesterol [(the enzyme method (CES4)-CO5)-POD6) method)], triglyceride [the enzyme method (LPL7)-GK8)-GPO9)-POD6) method)], total bilirubin (the enzyme method), urea nitrogen (the urease-UV method), creatinine (Jaffe method), AST (JSCC10) method), ALT (JSCC10) method) γ-GTP (JSCC10) method), ALP (JSCC10) method), LDH (SFBC11) method), calcium (OCPC12 method) and inorganic phosphorus [the enzyme method (PNP18)-XOD14)-POD6) method] were measured by using an automated biochemical analyzer (JCA-BM8, Clinalyzer, JEOL Ltd.), and sodium, potassium and chloride (the ion electrode method for these items) were measured by using an automated electrolyte analyzer (NAKL-132, DKK-TOA Corporation).
Note of the measurement methods; 1): bromcresol green, 2): glucokinase, 3): glucose-6-phosphate dehydrogenase, 4): cholesterol esterase, 5): cholesterol oxidase, 6): peroxidase, 7): lipoprotein lipase, 8): glycerol kinase, 9): L-α-glycerophosphate oxidase, 10): Japan Society of Clinical Chemistry, 11): Society of Clinical Biology (France), 12): o-cresolphthalein complexone, 13): purine-nucleoside phosphorylase, 14): xantine oxidase

Sacrifice and pathology:

Necropsy
Animals were exsanguinated and euthanized after blood sampling on the day following the end of treatment period and on the day following the end of recovery period. Then, the body surface, mucous membrane of the orifices and internal organs were macroscopically observed.

Organ weight
The brain, thymus, heart, liver, kidney, adrenal gland, spleen, thyroid gland and pituitary gland were weighed (absolute weight). Furthermore, the testis, epididymis, prostate gland and seminal gland (including the coagulating gland) in males and the ovary and uterus in females were also weighed (absolute weight). Then, the organ to body weight ratios (relative weight) were calculated based on the body weight measured on the day of necropsy. Paired organs were weighed together, and the thyroid gland, pituitary gland, prostate gland and seminal gland were weighed after being fixed with 10% neutral phosphate buffered formalin solution.

Histopathology
The following organs were collected from all animals, fixed with 10% neutral phosphate buffered formalin solution (the testis and epididymis were pre-fixed with Buin’s solution) and stored. The carcasses were incinerated at 800C or higher:
Brain, pituitary gland, eye ball, thyroid gland (including parathyroid), spinal cord (cervical, thoracic and lumbar parts), heart, aorta (thoracic part), trachea and lung (immersed in the fixative after injection of the fixative), liver, kidney, thymus, spleen, adrenal gland, tongue, esophagus, salivary gland (sublingual gland, submandibular gland, parotid gland), pancreas, stomach (forestomach and glandular stomach), small intestine [duodenum, jejunum, ileum (including Payer’s patch)], large intestine (cecum, colon and rectum), reproductive organ (testis or ovary), accessory reproductive organ (uterus, vagina or prostate gland, seminal gland, epididymis), urinary bladder, ischiatic nerve and the surrounding muscle, lymph node (cephalic lymph node, mesenteric lymph node), bone/bone marrow (sternum, femur), skin (abdominal region), mammary gland (abdominal region)
Histopathological examinations were conducted for the following organs from animals of the control group and the 0.5% treatment group: Brain (including cerebrum, cerebellum, medullary substance, pons cerebelli), pituitary gland, eye ball, thyroid gland, parathyroid gland, salivary gland (sublingual gland, submandibular gland, parotid gland), thymus, trachea, lung, heart, aorta (thoracic region), esophagus, stomach (forestomach and glandular stomach), small intestine [duodenum, jejunum, ileum (including Payer’s patch)], large intestine (cecum, colon and rectum), liver, pancreas, spleen, kidney, adrenal gland, urinary bladder, testis, prostate gland, seminal gland, epididymis, ovary, uterus, spinal cord (cervical, thoracic and lumbar parts), ischiatic nerve, skeletal muscle, bone marrow (femur), lymph node (cephalic lymph node, mesenteric lymph node), skin, mammary gland (female)
Paraffin sections of these organs were prepared, H-E stained and observed under microscopy according to the routine method. Histopathological examinations were not performed for the 0.056% and 0.167% treatment groups since no changes related to the test article were observed in the 0.5% treatment group. 

Statistics:

The mean and standard deviation were obtained for the parametric data [body weight, body weight gain, food consumption, urinalysis (urine volume, specific gravity, urinary sodium excretion, and urinary potassium excretion), hematological data, blood biochemical data and organ weight] in each group. When there were 3 study groups or more, the Bartlett’s variance test was conducted. When the homogeneity of variance assumption was met, then one-way ANOVA was conducted. When the homogeneity of variance assumption was not met, or for the non-parametric data (differential leukocyte counts, urinalysis), Kruskal-Wallis rank test was conducted. As a result, when there was a statistically significant difference, multiple comparison was made using Dunnett’s test or Dunnett-type test. When there were 2 study groups, F-test was conducted for the parametric data. When the homogeneity of variance assumption was met, Student’s t test was conducted. When the homogeneity of variance assumption was not met, Aspin-Welch’s t test was conducted. Mann-Whitney U test was conducted for the non-parametric data. Categorical data (incidences of animals with abnormal findings in the clinical observations, close clinical observations, sensorimotor function observations, necropsy, and histopathological examinations) were analyzed by using Fisher’s exact test. Histopathological data that were classified by grade level were analyzed by using Mann-Whitney U test. The significance level was 5% for all tests.

Clinical signs:

no effects observed

Description (incidence and severity):

No changes in clinical signs were observed in both male and female animals throughout the treatment period.
Loss of the right maxillary incisor was observed in one (Animal No. 547) of 5 females in the 0.5% treatment group as an incidental finding that was not related to treatment with the test article from Day 10 of recovery during the recovery period.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in both male and female animals throughout the treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant changes in body weight at each measuring time point and body weight gain during the treatment and recovery periods were observed in both male and female animals throughout the treatment and recovery periods.
Body weight of a female animal (Animal No. 547) in the 0.5% treatment group that had a loss of the right maxillary incisor on Day 10 of recovery decreased on Day 14 of recovery as compared to Day 7 of recovery.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During the treatment period, significantly low food consumption was observed in female animals of the 0.056% treatment group at Weeks 5 and 13 of treatment and significantly high food consumption was observed in female animals of the 0.5% treatment group at Weeks 3 and 11 of treatment. No significant changes were observed in male animals at any measuring time points. During the recovery period, low food consumption was observed in some female animals including one that had a loss of the right maxillary incisor in the 0.5% treatment group at Week 2 of recovery, and a significant difference was observed. No significant changes were observed in male animals. The intake of the test article (mean of each group from Week 1 of treatment to Week 13 of treatment) was 41 mg/kg/day in males and 46 mg/kg/day in females of the 0.056% treatment group, 120 mg/kg/day in males and 150 mg/kg/day in females of the 0.167% treatment group, and 380 mg/kg/day in males and 440 mg/kg/day in females of the 0.5% treatment group.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmological abnormalities were observed in both male and female animals in the tests at Week 13 of treatment and Week 2 of recovery.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly low reticulocyte counts were observed in male animals of the 0.056% and 0.5% treatment groups at the end of treatment period, but there was no dose-response relationship. No significant changes in any test items were observed in female animals.
Significantly low white blood cell counts in males and significantly shortened activated partial thromboplastin time and significantly high monocyte counts of the differential leukocyte counts in females were observed in the 0.5% treatment group at the end of the recovery period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significant decreases in total bilirubin in males and significant increases in γ-GTP and significant decreases in albumin, A/G ratio, triglyceride and total bilirubin in females were observed in the 0.056% treatment group at the end of treatment period. Significant decreases in total bilirubin in males and significant increases in total protein and albumin in females were observed in the 0.5% treatment group. No dose-response relationship was observed except for increased total protein levels due to increased albumin levels in males of the 0.5% treatment group.
Significant increases in inorganic phosphorus were observed in females of the 0.5% treatment group at the end of recovery period.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly high values of sodium, potassium and pH in the 0.056% treatment group, significantly high values of urine specific gravity, sodium, potassium and pH in the 0.167% treatment group and significantly high values of urine volume and sodium in the 0.5% treatment group were observed at Week 12 of treatment. However, no dose-response relationship with changes in urine specific gravity, sodium, potassium and pH was observed. No significant changes in any test items were observed in female animals.
No significant changes in any test items were observed in both male and female animals at Week 2 of recovery.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Close clinical observations
No changes related to treatment with the test article in any observation items were observed in both male and female animals throughout the treatment period and the recovery period.

Sensorimotor function observations
No abnormal reaction was observed in any tests in both male and female animals at Week 13 of treatment (at the end of treatment period) and Week 2 of recovery (at the end of recovery period).

Grip strength
No significant changes in forelimb and hindlimb grip strength were observed in both male and female animals in the tests at Week 13 of treatment and Week 2 of recovery.

Locomotor activity
A significantly high locomotor activity was observed in female animals of the 0.167% treatment group in the test at Week 13 of treatment, but there was no dose-response relationship with the change. No significant changes were observed in male animals of any test article treatment groups and female animals of the 0.056% and 0.5% treatment groups.
No significant changes were observed in both male and female animals in the tests at Week 2 of recovery.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly increased relative liver weight was observed in males and significantly increased relative kidney weight in females were observed in the 0.5% treatment group at the end of treatment period.
Significantly increased relative weights of the prostate gland and seminal gland were observed in males and significantly increased absolute and relative weights of the thyroid gland were observed in females of the 0.5% treatment group at the end of recovery period.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No changes related to treatment with the test article were observed.

As findings not related to treatment with the test article, accumulation of foam cells in the lung (female), degeneration/fibrosis of the myocardium (male and female), focal necrosis in the liver (male and female), micro-granuloma (male and female) and local biliary proliferation (male), basophilic tubule (male and female), cortical lymphocytic infiltration (female) and mineral deposition at the cortico-medullary junction (female) in the kidney, cyst in the anterior lobe of the pituitary gland (male), pharyngeal pouch remnant in the thyroid gland (female) and interstitial lymphocyte infiltration in the prostate gland were observed at a low incidence in the control and the 0.5% treatment groups. In addition, hyaline droplet in the proximal tubular epithelium in the kidney (male) and splenic extramedullary hematopoiesis and brown pigmentation (male and female) were observed at a high incidence; however there was no significant difference in the incidence and the severity between the control group and the 0.5% treatment group. In the 0.5% treatment group, eosinophilic body in the proximal tubular epithelium and unilateral infarction-like lesion with fibrosis and cyst-like dilated tubules in the kidney was also observed in one male animal of the 0.5% treatment group; however, renal infarction is observed in aged rats2) and it was considered to be an incidental finding that was not related to treatment with the test article.
Reduced size of the testis observed in male animals in the necropsy without any relation to treatment with the test article was confirmed to be caused by atrophy of seminiferous tubules, and uterine cavity edema that was also observed in the necropsy was confirmed to be a simple dilation of the uterine cavity. 2 of 3 male animals which had atrophy of seminiferous tubules had also decreases in sperm in the epididymis.

Histopathological findings: neoplastic:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

0.5 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Overall effects

Remarks on result:

other: Actual dose recieved: 380 mg/kg bw/d (male), 440 mg/kg bw/d (female)

Key result

Critical effects observed:

no

Discusstion

A 90-day dietary repeated-dose toxicity study of AMPHITOL 20HD was conducted in rats at a concentration of 0.056%, 0.167% and 0.5%.

No significant changes in clinical signs, mortality, close clinical observations, sensorimotor function observations and grip strength were observed. No ophthalmological changes related to treatment with the test article were observed.

Significantly high locomotor activity observed in female animals of the 0.167% treatment group was considered to be an incidental change without any relation to treatment with the test article since it was observed only in female animals without any dose-response relationship, and all the values were lower than the upper limit (19,229 counts/60 minutes) of our laboratory’s background data.

No significant changes in body weight at each measuring time point and body weight gain during the treatment were observed. As for food consumption, significantly low food consumption at Weeks 5 and 13 of treatment in the 0.056% treatment group and significantly high food consumption at Weeks 3 and 11 of treatment in the 0.5% treatment group were unlikely caused by treatment with the test article since there was no consistency of changes among the measuring time points, changes were occasionally observed, and no effect on body weight was observed.

There was no dose-response relationship with changes in urine specific gravity, sodium, potassium and urinary pH. Significantly increased urinary volume observed in males of the 0.5% treatment group was also a change within the reference values except for the value in one of 10 animals that slightly exceeded the upper limit (19.8 mL/18 hours) of the reference value of our laboratory’s background data. There were no changes in serum electrolyte levels and there were also no relevant changes in blood biochemistry and histopathological examination. Therefore, changes observed in the urinalysis were considered to be incidental changes without any relation to treatment with the test article.

Significant decreases in reticulocyte counts observed in only male animals of the 0.056% treatment group and the 0.5% treatment group were also considered not to be changes indicative of the effect of the test article on red blood cells since there was no dose-response relationship.

Significantly high total protein levels caused by high albumin levels were observed in females of the 0.5% treatment group in the blood biochemistry. However, there was no dose-response relationship with high albumin levels, and the total protein level in each animal was lower than the upper limit (7.31 g/dL) of the reference value of the background data.

In addition, there was also no dose-response relationship with significantly low total bilirubin levels that were occasionally observed in male animals and significantly high γ-GTP and A/G ratio and significantly low levels of triglyceride and total bilirubin that were observed in female animals in each treatment group. Changes in total bilirubin levels were considered not to be related to increased relative liver weight in male animals described later since total bilirubin levels tended to decrease and these changes were not indicative of liver disorder. Thus, all of the blood biochemical changes observed in this study were considered not to be indicative of a toxic effect of the test article.

As for organ weights, the test article was considered to likely have some effects on the liver and kidney, since significantly increased relative weight of the liver (male) and kidney (female) was observed in the 0.5% treatment group, a slightly similar tendency was also observed for relative weight of the liver (female) and kidney (male) and a slight tendency of dose-related changes was also observed. However, there were no histopathological changes related to treatment with the test article in the liver and kidney or blood biochemical changes indicative of impairment of liver function and renal function. No adverse effect of the test article on the liver and kidney was observed.

No histopathological changes related to treatment with the test article were also observed in the other organs.

Thus, no adverse effects caused by treatment with the test article were observed although significantly increased relative weight of the liver (male) and kidney (female) was observed in the 0.5% treatment group during the treatment period and at the end of treatment period. Changes in these weights of the liver and kidney were not observed at the end of recovery period, reversible and returned to the baseline level in a short term.

Changes in food consumption, hematological changes, blood biochemical changes and changes in organ weight observed in the recovery groups were considered to be incidental changes that were not indicative of a delayed toxic effect of the test article since these changes were not observed at the end of treatment period, there was no consistency between sexes of animals, and there were no accompanied changes.

From these results, no changes related to treatment with the test article were observed in the 0.056% treatment group (Intake of the test article was 41mg/kg/day in males and 46 mg/kg/day in females) and the 0.167% treatment group (Intake of the test article was 120 mg/kg/day in males and 150 mg/kg/day in females) in a 90-day dietary repeated-dose toxicity study of AMPHITOL 20HD. No adverse effect of the test article on the liver and kidney was observed although slightly increased relative weight of liver (male) and kidney (female) was observed in the 0.5% treatment group (Intake of the test article was 380 mg/kg/day in males and 440 mg/kg/day in females). Thus, it was concluded that the no-observed adverse effect level (NOAEL) was 0.5% or more in both male and female animals.

Conclusions:

The results indicate that there was no change attributable to the test article in the 0.056% group (the intake of the test article was 41 mg/kg/day in males and 46 mg/kg/day in females) and 0.167% group (120 and 150 mg/kg/day, respectively) after the 90-day dietary administration of Amphitol 20HD in rats. In the 0.5% group (380 and 440 mg/kg/day, respectively), the relative weight of the liver in males and that of the kidney in females was slightly increased, although there was no adverse effect on the liver or kidney. It is therefore concluded that the no-observed adverse effect level (NOAEL) was not less than 0.5% for both male and female rats.

Executive summary:

A 90 -day dietary repeated-dose toxicity study of AMPHITOL 20HD was conducted in rats at concentrations of 0%, 0.056%, 0.167% and 0.5%. No significant changes in clinical signs, mortality, close clinical observations, sensorimotor function observations and grip strength were observed. No ophthalmological changes related to treatment with the test article were observed. Significantly high locomotor activity observed in female animals of the 0.167% treatment group was considered to be an incidental change without any relation to treatment with the test article since it was observed only in female animals without any dose-response relationship, and all the values were lower than the upper limit (19,229 counts/60 minutes) of the laboratory’s background data. No significant changes in bodyweight at each measuring time point and bodyweight gain during the treatment were observed. As for food consumption, significantly low food consumption at Weeks 5 and 13 of treatment in the 0.056% treatment group and significantly high food consumption at Weeks 3 and 11 of treatment in the 0.5% treatment group were unlikely caused by treatment with the test article since there was no consistency of changes among the measuring time points, changes were occasionally observed, and no effect on body weight was observed. There was no dose-response relationship with changes in urine specific gravity, sodium, potassium and urinary pH. Significantly increased urinary volume observed in males of the 0.5% treatment group was also a change within the reference values except for the value in one of 10 animals that slightly exceeded the upper limit (19.8 mL/18 hours) of the reference value of the laboratory’s background data. There were no changes in serum electrolyte levels and there were also no relevant changes in blood biochemistry and histopathological examination. Therefore, changes observed in the urinalysis were considered to be incidental changes without any relation to treatment with the test article. Significant decreases in reticulocyte counts observed in only male animals of the 0.056% treatment group and the 0.5% treatment group were also considered not to be changes indicative of the effect of the test article on red blood cells since there was no dose-response relationship. Significantly high total protein levels caused by high albumin levels were observed in females of the 0.5% treatment group in the blood biochemistry. However, there was no dose-response relationship with high albumin levels, and the total protein level in each animal was lower than the upper limit (7.31 g/dL) of the reference value of the background data. In addition, there was also no dose-response relationship with significantly low total bilirubin levels that were occasionally observed in male animals and significantly high γ-GTP and A/G ratio and significantly low levels of triglyceride and total bilirubin that were observed in female animals in each treatment group. Changes in total bilirubin levels were considered not to be related to increased relative liver weight in male animals described later since total bilirubin levels tended to decrease and these changes were not indicative of liver disorder. Thus, all of the blood biochemical changes observed in this study were considered not to be indicative of a toxic effect of the test article. As for organ weights, the test article was considered to have some effects on the liver and kidney, since significantly increased relative weight of the liver (male) and kidney (female) was observed in the 0.5% treatment group, a slightly similar tendency was also observed for relative weight of the liver (female) and kidney (male) and a slight tendency of dose-related changes was also observed. However, there were no histopathological changes related to treatment with the test article in the liver and kidney or blood biochemical changes indicative of impairment of liver function and renal function. No adverse effect of the test article on the liver and kidney was observed. No histopathological changes related to treatment with the test article were also observed in the other organs. Thus, no adverse effects caused by treatment with the test article were observed although significantly increased relative weight of the liver (male) and kidney (female) was observed in the 0.5% treatment group during the treatment period and at the end of treatment period. Changes in these weights of the liver and kidney were not observed at the end of recovery period, reversible and returned to the baseline level in a short term. Changes in food consumption, hematological changes, blood biochemical changes and changes in organ weight observed in the recovery groups were considered to be incidental changes that were not indicative of a delayed toxic effect of the test article since these changes were not observed at the end of treatment period, there was no consistency between sexes of animals, and there were no accompanied changes. From these results, no changes related to treatment with the test article were observed in the 0.056% treatment group (41 mg/kg bw/d in males and 46 mg/kg bw/d in females) and the 0.167% treatment group (120/150 mg/kg bw/d) in a 90-day dietary repeated-dose toxicity study of AMPHITOL 20HD. No adverse effect of the test article on the liver and kidney was observed although slightly increased relative weight of liver (male) and kidney (female) was observed in the 0.5% treatment group (380/440 mg/kg bw/d). Thus, it was concluded that the no-observed adverse effect level (NOAEL) was 0.5% in both male and female animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

380 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Study has been conducted to GLP in accordance with OECD guidelines.

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

Justification for classification or non-classification

The results of the 90 -day toxicity study do not trigger classification (STOT-RE) under CLP.