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Description of key information

Oral, 28 days, rat: NOAEL (systemic) = 150 mg/kg bw/day (similar to OECD 407)

Oral, 90 days, rat: NOAEL (systemic) = 150 mg/kg bw/day (similar to OECD 408)

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:
May 9 to June 29 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: Section 4 of the Guidelines for the Testing of Chemicals: Health Effects (ministry of environmental protection of People's Republic of China)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted Sept. 1998
Deviations:
yes
Remarks:
limited documentation (no raw data or results from individual animals were included in the study report), no food consumption measured, no detailed clinical observations, no ophthalmological examination
GLP compliance:
not specified
Remarks:
study was performed in China and it is not clear whether it was performed according to GLP
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat was the preferred rodent species for this study and the SD strain was chosen due to its sensitivity against the known chemicals and uniformity in genetic properties.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Shanghai SLAC Laboratory Animal Co., Ltd.
- Age at study initiation: not specified
- Weight at study initiation: 110 g to 140 g
- Fasting period before study: no
- Housing: 2 per polycarbonate cage
- Diet: powdered diet supplied by Suzhou Shuangshi Laboratory Animal Feed Co., Ltd., study report does not specify if feed was provided ad libitum
- Water: ad libitum
- Acclimation period: five days

DETAILS OF FOOD AND WATER QUALITY: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Prescribed amount of the test substance was weighed with a balance, and then diluted with corn oil to the specified concentrations.

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of an acute oral toxicity and repeated dose 28-day oral toxicity study.
- Rationale for selecting satellite groups: additional high dose and control group were assigned to assess the effects of a 14-day recovery period
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: not specified
- Cage side observations: general clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: not reported
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: not reported

FOOD EFFICIENCY:
- Food efficiency in percent is given in the results, no further description how these were dervived are given in the report

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the administration period (after 90 days)
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Not specified
- How many animals: all the animals in the control and treatment groups
- Parameters checked: red blood cell (RBC), hemoglobin (Hb) content, white bood cell (WBC), leukocyte diffierential count (the rate of neurocytes (NEU%), the rate of lymphocytes (LYM%), the rate of monocytes (MONO), the rate of basophils (BASO%), the rate of eosinophils (EOS%)), platelet (PLT) counts, plasma prothrombin time (PT) and activated partial thromboplatin (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period
- Animals fasted: Not specified
- How many animals: all
- Parameters checked: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cholesterol total (CHOL), bilirubin total (TBIL), glucose(GLU), blood urea nitrogen (BUN), creatinine (CREA), cholinesterase (CHE), total protein (TP), albumin (ALB), Na, K, Cl, Ca and P ions

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of administration period
- Metabolism cages used for collection of urine: not specified
- Animals fasted: not specified
- Parameters checked: specific gravity, color, pH, protein contents, blood/blood cells, glucose concentration

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: All the rats were subjected to a full gross necropsy and macroscopically observed in detail.
The following organs were weighed: brain, heart, lung, liver, spleen, kidney, adrenal gland, testis or ovary and thymus

HISTOPATHOLOGY: Yes: brain, heart, lung, liver, thymus, spleen, kidney, adrenal gland, testis or ovary, vervus ischiadicus, cervical, spinal cord, thyroid, pancreas, stomach, duodenum, jejunum, ileum, colon, rectum, mesenteric lymph node, prostate, epididymides, urinary bladder and uterus
Statistics:
Data were analyzed by ANOVA, and the additional high and control dosage group were analyzed by t-test using SPSS10.0.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Compared with the control group, the body weight gain of male rats in the high dose group was significantly decreased from the first (-6.4%) to the sixth (-8.6%) week and the eighth (-8.7%) to the thirteenth (-10.0%) week (p<0.05). The body weight gain of male rats in the high dose recovery group was significantly decreased during the entire treatment and recovery period, compared with the recovery control group (-4.5% in the first week up to -10.8%) in the fifteenth week. No effects were noted on body weight (gain) in the females of any treatment group, compared with the control group.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematology analysis showed that red blood cell count of male rats in the high dose group was significant lower than in control group (-7.2%). The red blood cell counts were not significantly different in the high dose recovery group compared to the control recovery group, showing that this effect is reversible and thus probably not adverse. No historical reference data are given in the report but comparing the values for the red blood cell count in male rats of the high dose group (8.35 x 10E12/L) with historical reference data from male Wistar rats* ((7.37 - 9.25 x 10E12/L; age 19 - 21 weeks) the red blood cell counts of male rats in the high dose group falls within this historical range supporting that this effect is not of toxicological relevance. Basophil levels of female rats in the high dose recovery group were significantly lower than in the control group (-38%). Since the overall white blood cell count is not affected this effect is likely to be incidental and not treatment-related. Furthermore, the basophil levels (0.42%) fall in the range of historical reference values of female Wistar rats* (0 - 2%; age 19 - 21 weeks).

*technical bulletin: Baseline Hematology and Clinical Chemistry Values for Charles River Wistar Rats - (CRL:(WI)BR) as a Function of Sex and Age (http://www.criver.com/files/pdfs/rms/wistar-rats/rm_rm_r_hematology_crl_wi_br_sex_age.aspx)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Female rats: The cholinesterase level was significantly decreased (-33.7% in high dose group, -20.4% in high dose recovery group) in the high dose group and high dose recovery group compared to the respective control groups. However, no correlation in liver weights or histopathology was found in female rats. But in male rats effects on liver weights and histopathology was observed which indicates a treatment-related effect but cannot clearly be defined as toxicological relevant.
The calcium level was significantly increased (+7.6% in the high dose group, +6.5% in the high dose recovery group) in the high dose group and high dose recovery group compared with the respective control groups. This effect is considered to be incidental since it is only a minimal change. The blood urea nitrogen (BUN, +27.4%) was significantly increased and the cholesterol levels (-15.2%) were significantly decreased in the high dose recovery group compared to the control recovery group. This effect is considered to be not toxicological relevant since it only occurred in the recovery group. Chloride was significantly increased in the high dose group (+2.6%) but not in the high dose recovery group, thus indicating a reversible effect.
Male rats: Aspartate aminotransferase was significantly decreased (-14.3% in mid dose, 14.7% in high dose group), and cholinesterase was significantly increased (+48.3% in mid dose, +78.4% in high dose group) in the mid- and high dose groups, compared with the respective control groups. For both these effects, no significant difference was observed between the recovery treatment group and control group, indicating the effect was reversible. Creatinine was significantly increased (+26.7%) in the mid dose group, compared with the control group. As no similar effect was noted in the high dose group, this is considered to be incidental.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
One male rat each in the high and the control group showed a positive reaction in the protein measurement of the urine. This effect is considered to be incidental and not treatment-related since it was seen in the high dose group as well as in the control group.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males: The liver weight (-22.1%) was significantly reduced in the high dose recovery group compared to the control group. The relative liver weight was significantly increased for the high dose group (+14.2%) and significantly reduced for the high dose recovery group (-13.2%) compared to the respective control groups. The effects observed in the liver are considered to be treatment-related.
The relative organ weight of the brain was significantly higher in the high dose (+11.8%) and high dose recovery group (+12.1%) than in the respective control groups. Relative heart (+11.1%) and kidney (+17.1%) weight increases were observed in the high dose group of male rats compared to the control group.
Relative organ weight increase in testes are observed in the high (+12.1%) and mid (+10.3%) dose group and the high dose recovery group (+15.8%) of male rats compared to the respective control group. Since the body weight was significantly reduced in male rats of the high dose group and the organ weights did not change these effects are secondary due to the reduced body weight.
The spleen weight (-22.7%) and the relative spleen weight (-16.7%) was significantly reduced in the high dose recovery group. The effect seen in the spleen is considered to be incidental since it is only observed in the recovery group.

Females: In the mid dose group (150 mg/kg bw/day) for female rats the kidney weight was significantly lower (-10.3%) than for the control group. No effects were observed in the recovery group or in the relative kidney weight, therefore this effects is considered incidental.
The relative organ weight of adrenal gland was significantly higher (+26%) in the mid dose group of female rats compared to the control group. Since no dose-response was observed this effect is considered incidental.
The ovary weight (+45.5%) and the relative ovary weight (+25%) of female rats in high dose recovery group were significantly increased compared to the control recovery group. Since this effect was only observed in the recovery group this effect is considered incidental.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following histopatholgogical findings were observed: Two case in females of epithelial cells degeneration of kidney tubules, seven cases in males rats of infiltration of inflammatory cells in portal area and fatty degeneration of liver cells. The effects observed in the kidney cannot clearly be defined as toxicologically relevant or treatment-related, due to limited information in the study report. The histopathologic findings in the liver are probably treatment-related since they are found in a high number of male rats (7/10) in the high dose group.
No overt abnormality was found in the middle, low and recovery groups.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: urinary and hepatobiliary
Organ:
liver
kidney
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
Oral application of 1000, 150 and 25 mg/kg bw/day test substance for 90-days to male and female Sprague-Dawley rats resulted in a significant body weight decrease in male rats of the high dose group, organ to body weight change in the liver of the high dose group in male rats and histopathology changes in liver in male rats and kidney in female rats of the high dose group. Based on these results a NOAEL is set to 150 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.
System:
other: hepatobiliar and urinary
Organ:
liver
kidney

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

Mode of Action Analysis / Human Relevance Framework

not applicable

Additional information

Subacute toxicity:

The subacute repeated dose toxicity study was conducted similar to OECD 407. In this study, groups of 5 male and 5 female rats received PEMB in olive oil orally via gavageat dose levels of 25, 150, 1000 mg/kg/ bw/day for 28 days (Miyata, 2009). In addition, satellite groups for the vehicle control and the top dose were included for follow-up observations. After the testing period, the animals of the satellite groups were kept 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

During treatment no animals died. Clinical signs observed were considered to be not treatment-related, as they occurred spontaneously. Moreover, there was no evidence for a clear dose-response relationship. During recovery, no abnormalities were noted for clinical signs, except of plenty of food on the tray. However, as the vehicle was olive oil it is conceivable that the animals were not hungry based on increased caloric intake due to the vehicle. Therefore, clinical signs observed were considered as incidental findings among control and treated animals.

No statistically significant increases of body weights were noted in all treated females. In males, body weights were significantly decreased in the 1000 mg/kg bw/day group after day 21 of treatment. However, this effect on body weight was reversible during recovery.

For the females, locomotor activity counts were spontaneously increased during treatment. In females of the 1000 mg/kg bw/day recovery group, no abnormal locomotor activity counts were detected. In males, no effects were noted both during treatment and recovery.

At termination of treatment, relative liver weights of both sexes were increased in the high dose animals. Absolute liver weights were only increased in females. Enlargement of the liver was observed in 1 of 5 females of the 150 mg/kg bw/day group and in all females of the 1000 mg/kg bw/day group. In males, enlargement of the liver was found in 2 of 5 animals of the high dose group. Histopathological examinations of the high dose group showed diffuse fatty change of the hepatocytes in males and fatty changes of the periportal hepatocytes in females. The abnormal findings in the liver of males and females were also reflected in the blood chemistry data: increased total level of bilirubin in both sexes, increased activity of alanine aminotransferase in males and decreased activity of cholinesterase in females at 1000 mg/kg bw/day.

At the end of the recovery phase, relative liver weights were significantly increased in males in the high dose group as shown at the end of treatment. However, the severity was decreased compared to those at the end of treatment. Liver did not show significant changes of organ weights in females at the end of the recovery period. No histopathological changes were found in the liver of males and females, indicating that histopathological findings observed after treatment were reversible. It should be noted that male rats are susceptible to chemically induced renal toxicity involving alpha-2µ-globulin, therefore, it is not clear whether the effect on the male liver is mediated by alpha-2µ-globulin.

In the kidneys, relative weights in both sexes and absolute weights in females were increased in the 1000 mg/kg bw/day group. Also enlargement of the kidneys was found in both sexes in the high dose group. Degeneration and necrosis of the tubular epithelium with the regenerative epithelium was noted only in the high dose females.

At the end of the recovery phase, absolute and relative kidney weights were significantly increased in females in the high dose group as shown at the end of treatment. However, the severities were decreased compared to those at the end of treatment. Kidneys did not show significant changes of organ weights in males at the end of the recovery period. In the histopathological examination, no abnormal findings were observed in the kidney of males and females after the recovery period of 14 days. This means that the histopathological findings observed during treatment are reversible effects.

Based on the results of the study, the NOAEL for PEMB was considered to be 150 mg/kg bw/day due to overall effects on males and females at 1000 mg/kg bw/day.

Subchronic toxicity:

The subchronic repeated dose toxicity study was conducted according to a protocol similar to OECD guideline 408. The deviations include limited documentation: no raw data results or results from individual animals were included in the study report.  Furthermore, it was not specified if the food consumption was measured, and no detailed clinical observations or ophthalmological examinations were performed. In this study, groups of 10 male and 10 female Sprague Dawley rats received PEMB in corn oil orally via gavage at dose levels of 25, 150, 1000 mg/kg/ bw/day for 90 days (Jun, 2010). In addition, recovery groups for the vehicle control and the high dose were included. After the treatment period, the animals of the recovery groups were kept 14 days without treatment in order to detect delayed occurrence of, or persistence of, or recovery from toxic effects. General clinical signs were recorded, and body weight, food efficiency, organ weights, hematology parameters, clinical biochemistry parameters, and urinary parameters were measured. All animals were subjected to a full gross necropsy and macroscopically observed in detail after the treatment period. Tissues from control and high dose groups were histologically examined.

No mortality was observed during the treatment, and no obvious clinical signs were observed during the study. Compared with the control group, the body weight gain of male rats in the high dose group was significantly decreased from the first (-6.4%) to the sixth (-8.6%) week and the eighth (-8.7%) to the thirteenth (-10.0%) week. The body weight gain of male rats in the high dose recovery group was significantly decreased during the entire treatment and recovery period, compared with the recovery control group (-4.5% in the first week up to -10.8%) in the fifteenth week. No effects were noted on body weight (gain) in the females of any treatment group, compared with the control group. The food efficiency was not significantly changed in either group.

The absolute liver weight in males (-22.1%) was significantly reduced in the high dose recovery group compared with the control group. The relative liver weight was significantly increased for the male high dose group (+14.2%) and significantly reduced for the male high dose recovery group (-13.2%) compared with the respective control groups. The effects observed in the liver are considered to be treatment-related, although the toxicological significance of the observed changes is unclear. All other significant changes in absolute or relative organ weights were considered to be incidental or secondary due to the decreased body weight in males and females during the study.

The hematology analysis showed that the red blood cell count of male rats in the high dose group was significant lower than in control group (-7.2%). The red blood cell counts were not significantly different in the high dose recovery group, compared with the control recovery group, indicating that this effect is reversible and most likely not adverse. No historical reference data are given in the report but comparing the values for the red blood cell count in male rats of the high dose group (8.35 x 10E12/L) with historical reference data from male Wistar rats* ((7.37 - 9.25 x 10E12/L; age 19 - 21 weeks) the red blood cell count of male rats in the high dose group falls within this historical range, supporting the conclusion that this effect has no toxicological relevance. Basophil levels of female rats in the high dose recovery group were significantly higher than in the control group (-38%). Since the overall white blood cell count is not affected this effect is likely to be an incidental effect. Furthermore, the basophil levels (0.42%) fall in the range of historical reference values of female Wistar rats* (0 - 2%; age 19 - 21 weeks).

The clinical biochemistry analysis showed the following results for female rats: The cholinesterase level was significantly decreased (-33.7% in high dose group, -20.4% in high dose recovery group) in the high dose group and high dose recovery group compared with the respective control groups. However, no correlation in liver weights or histopathology was found in female rats. But in male rats effects on liver weights and histopathology was observed which indicates a treatment-related effect but cannot clearly be defined as toxicological relevant.

The calcium level was significantly increased (+7.6% in the high dose group, +6.5% in the high dose recovery group) in the high dose group and high dose recovery group compared with the respective control groups. This effect is considered to be incidental since it is only a minimal change. The blood urea nitrogen (BUN, +27.4%) was significantly increased and the cholesterol levels (-15.2%) were significantly decreased in the high dose recovery group compared to the control recovery group. This effect is considered to be not toxicological relevant since it only occurred in the recovery group. The chloride level was significantly increased in the high dose group (+2.6%) but not in the high dose recovery group, thus indicating a reversible effect.

For male rats the following results were observed: Aspartate aminotransferase was significantly decreased (-14.3% in mid dose, 14.7% in high dose group), and cholinesterase was significantly increased (+48.3% in mid dose, +78.4% in high dose group) in the mid- and high dose groups, compared with the respective control groups. For both these effects, no significant difference was observed between the recovery treatment group and control group, indicating the effect was adaptive. Creatinine was significantly increased (+26.7%) in the mid dose group, compared with the control group. As no similar effect was noted in the high dose group, this is considered to be an incidental effect.

The routine urinalysis showed no abnormal results.

The gross necropsy did not show abnormalities in the animals. The histopathological analysis of female rats revealed epithelial cell degeneration of kidney tubules in 2/10 the high dose group. The effects observed in the kidney cannot clearly be defined as toxicologically relevant or treatment-related, due to limited information in the study report. For male rats in the high-dose group, there were seven cases of infiltration of inflammatory cells in portal area and fatty degeneration of liver cells. The histopathologic findings in the liver are probably treatment-related since they are found in a high number of male rats (7/10) in the high dose group. There was no overt abnormality in the middle and low dose groups.

Based on the results, the NOAEL of PEMB in this 90-day oral toxicity test in rats is considered to be 150 mg/kg bw/day.  

*technical bulletin: Baseline Hematology and Clinical Chemistry Values for Charles River Wistar Rats - (CRL:(WI)BR) as a Function of Sex and Age (http://www.criver.com/files/pdfs/rms/wistar-rats/rm_rm_r_hematology_crl_wi_br_sex_age.aspx)

Justification for classification or non-classification

The available data on repeated dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.