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Description of key information

Two subacute oral toxicity studies and one subchronic oral toxicity study were evaluated. The dose descriptor selected for the Chemical Safety Assessment is the NOAEL (3 mg/kg bw) deduced from the key subacute study. Only one dermal repeated dose toxicity study is available and was used for dermal - local and systemic effects

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to OECD guideline, GLP
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 1981-05-12
Deviations:
not specified
Qualifier:
according to
Guideline:
other: EC Guideline 84/449/EC, J. of Eur. Publ. L251, 1984-09-19
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: average 186.75 g for males, average 140.7 for females
- Housing: suspended stainless steel cages fitted with wire mesh floor and front, in groups of 5, separated by sex
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 1992-03-31 To: 1992-05-10
Route of administration:
oral: gavage
Vehicle:
other: diluted aqueous HCl, adjusted to pH c. 3.8 using NaOH
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The top dose dilution was prepared by dissolving 0.6 g Kerobit BPD in 30 mL 0.2N HCl. Then 160 mL demineralized water was added and the pH was adjusted to 3.8 using 4N NaOH. Finally the dilution was made up to 200 mL with demineralized water. The top dose thus contained 0.6 g Kerobit BPD per 200 mL diluted HCl. The lower dose dilutions were prepared by diluting the top dose solution with demineralized water. If necessary, the pH was adjusted to the same pH value as the top dose dilution with either 4N NaOH or 2N HCl. The demineralized water and the 0.3N HCl solutions were saturated with argon prior to use to supersede oxygen.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Spectra of solutions of Kerobit BPD were run on a Varian Cary 219 UV spectrometer from 700 nm to 200 nm.

The actual concentrations of the Kerobit BPD solutions are close to the nominal concentrations.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 3, 10 and 30 mg/kg
Basis:
other: nominal, dosed by gavage as a solution in diluted aqueous HCl.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected on the basis of a preceding oral (5-day) range-finding study with Kerobit BPD in rats (TNO-report v91.572, dated November, 1992 and amendment I to report v91.572, dated March, 1994)

data owner: BG Chemie (Berufsgenossenschaft Chemie)

data submitter has letter of access

Doses of 0, 10, 30 and 100 mg / kg body weight were administered daily for 5 consecutive days to groups of 5 male and 5 female rats. The test substances induced a number of changes in the top-dose group like a higher incidence of alopecia, growth retardation, in males associated with decreased food intake and food conversion efficiency and effects on red blood cell system, lymphocytes, clotting parameters and plasma clinical chemistry variables. Moreover in both sexes the relative liver weight was increased whereas the relative weight of the thymus was decreased in males. A number of these changes viz. effect on clotting parameters, clinical chemistry parameters and organ weights were also observed in the mid-dose group. Whether the intercurrent death of one female of the top-dose group has to be considered of toxicological significance is unclear since it can not be excluded that the cause of death is due to faulty dosing. Pathological examination of the animal (lungs) could not be performed anymore due to cannibalism.



Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily (2x on working days, 1x on weekend days)

CLINICAL OBSERVATIONS: All abnormalities, signs of ill health or reaction to treatment were recorded

BODY WEIGHT: Yes
- Time schedule for examinations: initially before the experiment and weekly thereafter

FOOD CONSUMPTION: Yes
- Food consumption for each cage was determined and mean daily diet consumption calculated as g food/rat/day

FOOD EFFICIENCY: Yes
- The efficiency of food utilization was calculated and expressed as gram weight gain per gram food consumed

WATER CONSUMPTION: Yes
- Time schedule for examinations: water bottles were weighted daily during day 1-7 (week 1) and during day 15-21 (week 3)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 25
- Animals fasted: Yes
- How many animals: 27
- Examined parameters: haemoglobin concentration, percentage methaemoglobin, packed cell volume, red blood cell count, red blood cell distribution width, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, reticulocyte count, Heinz body count, mean platelet volume, platelet distribution width, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 25 and day 28
- Animals fasted: Yes
- How many animals: 27
- Examined parameters: alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase acitivity, gamma glutamyl transferase activity, total protein, albumin, albumin/globulin ratio, urea, creatinine, bilirubin total, sodium (Na), potassium (K), calcium (Ca), chloride (Cl), inorganic phosphate, total cholesterol, triglycerides

URINALYSIS: No

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

All surviving rats were killed on day 28 in such a sequence that on the average the time of killing was about the same for each group. The animals were anaesthetized by ether, bled to death by cannulating the abdominal aorta and then examined grossly for pathological changes. Samples of the following tissues and organs of all animals were preserved in a neutral, aqueous, phosphate-buffered, 4% solution of formaldehyde: all gross lesions, adrenals, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroid (with parathyroids), urinary bladder, oesophagus and stomach.
The following organs of all surviving animals were weighed: adrenals, kidneys, liver, spleen, testes and thymus.
The tissues required for microscopic examination were embedded in paraffin wax, sectioned at 5 microm and stained with haematoxylin and eosin. Histological examination was performed on the liver, kidneys, spleen, adrenals and heart in all rats of the control group and the top-dose group.
Histopathological examination of the liver was extended to rats of mid- and low-dose groups because treatment-related microscopic changes were found in the top-dose group.
Statistics:
Body weights were evaluated by one-way analysis of co-variance followed by Dunnett's multiple comparison tests. Haematological variables (except for those mentioned below), clinical chemistry values and organ weights were evaluated by one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison tests. Differential white blood cell count, methaemoglobin and Heinz body and reticulocyte counts were analysed by Kruskal-Wallis nonparametric ANOVA followed by the Mann-Whitney U-test (for percentages). Histopathological changes were evaluated by Fisher's exact probability test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
increased incidence of focal alopecic areas and sparsely haired regions
Mortality:
mortality observed, treatment-related
Description (incidence):
increased incidence of focal alopecic areas and sparsely haired regions
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
increase in total white blood cell count, not dose-related and considered to be of no toxicological relevance
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
females of the mid and top-dose group: increased plasma concentrations of glucose and albumine
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver, kidney
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY:
Female rats of the top-dose group: increased incidence of focal alopecic areas and sparsely haired regions


FOOD CONSUMPTION:
Not significantly changed.


FOOD EFFICIENCY:
Not significantly changed.


WATER CONSUMPTION:
Top-dose group (male and female): slight, but consistent increase in water consumption


HAEMATOLOGY:
Females of the low and top-dose group: increase in total white blood cell count, not dose-related and considered to be of no toxicological relevance


CLINICAL CHEMISTRY:
Females of the mid and top-dose group: increased plasma concentrations of glucose and albumine
Females of the top-dose group: CGT activity and the concentrations of the total protein, bilirubin, cholesterol and calcium were increased
Males of the low-dose group showed an isolated decrease in bilirubin concentration. This was considered to be fortuitous because it was not found in the top-dose group.


ORGAN WEIGHTS:
Liver: absolute and relative weight was increased in males and females of the top-dose group; kidney: relative weight increased in females in the top dose group


HISTOPATHOLOGY: NON-NEOPLASTIC:
Microscopic examination revealed clear histopathological changes in the liver of the mid- and top-dose rats. These changes consisted of too homogenous cytoplasm (ground-glass cytoplasm) in the periportal hepatocytes. The incidence and the degree of severity were dose-related and the difference with the controls were statistically significant in the top-dose group.

Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The no-toxic effect level, as mentioned in the study report, is considered as the NOAEL.
Critical effects observed:
not specified
Conclusions:
Because no treatment-related changes were observed at the low-dose level, the no-toxic effect level of Kerobit BPD in this study was 3 mg/kg body weight/day.

Executive summary:

1. The oral toxicity of Kerobit BPD was examined in a 28-day oral toxicity study. The test substance was dissolved in a HCl solution and further diluted in demineralized water. The Kerobit BPD solutions were administered daily by gavage to groups of 5 male and 5 female rats for 28 consecutive days at dose levels of 0 (control), 3, 10 and 30 mg/kg body weight.

2. One male of the mid-dose group died on day 24 of the study, probably due to a dosing error and/or regurgitation and aspiration of the dosing solution. The death of this animal was not ascribed to any toxicity of the test substance. An increased incidence of focal alopecic areas was observed in females of the top-dose group. Otherwise, there were no relevant differences in appearance or behaviour between the treated rats and the controls.

3. Body weights and food and water intake were not significantly affected by the treatment.

4. The total number of white blood cells was increased in females of the low and top-dose group, which was mainly due to an increase in lymphocytes. This effect was not dose-related.

5. Clinical chemistry showed increased glucose and albumine levels in the plasma of females in the mid- and top-dose groups. Plasma gamma-glutamyltransferase (GGT) activity and concentrations of the total protein, bilirubin, cholesterol and calcium were significantly increased in females of the top-dose group.

6. At the end of the treatment the absolute and relative weight of the liver were increased in the top-dose group of both sexes. In addition, the relative weight of the kidney was increased in females in the top dose group.

7. There were no treatment-related gross abnormalities. Upon microscopy, hepatocellular alterations in the periportal area of the liver (ground glass cytoplasm) was observed in the mid- and top-dose rats in both sexes. The incidence and severity of these alterations was treatment-related.

8. Because no treatment-related changes were observed at the low-dose level, the no-toxic effect level of Kerobit BPD in this study was 3 mg/kg body weight / day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
3 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only secondary source available. No data on test protocol.
Limit test:
yes
Species:
rat
Strain:
not specified
Sex:
male
Type of coverage:
not specified
Vehicle:
not specified
Details on exposure:
clipped dorsal skin
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
5 days / week
Remarks:
Doses / Concentrations:
90 mg / kg bw
Basis:
nominal per unit body weight
No. of animals per sex per dose:
3 male rats
Control animals:
yes
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
irritant effects and ulceration of the skin
Mortality:
no mortality observed
Details on results:
No changes in haematological parameters other than granulocytosis were observed in the treated rats compared with 3 control rats.
The methaemoglobin level was not increased.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
90 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Only one dermal repeated dose toxicity study available, Klimisch score 4.
System:
other: Digestive
Organ:
liver

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only secondary source available. No data on test protocol.
Limit test:
yes
Species:
rat
Strain:
not specified
Sex:
male
Type of coverage:
not specified
Vehicle:
not specified
Details on exposure:
clipped dorsal skin
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
5 days / week
Remarks:
Doses / Concentrations:
90 mg / kg bw
Basis:
nominal per unit body weight
No. of animals per sex per dose:
3 male rats
Control animals:
yes
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
irritant effects and ulceration of the skin
Mortality:
no mortality observed
Details on results:
No changes in haematological parameters other than granulocytosis were observed in the treated rats compared with 3 control rats.
The methaemoglobin level was not increased.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
Only one dermal repeated dose toxicity study available, Klimisch score 4.

Additional information

Subacute oral toxicity

The subacute oral toxicity of 44 PD was evaluated in two 28-days repeated dose-toxicity studies (Groten, J.P., 1994 (key) and Shellenberger, T.E., 1984, (supporting)).

In the key study, the test substance was dissolved in a diluted aqueous HCl and this solution was administered to male and female Wistar rats. Doses of 0, 3, 10 and 30 mg/kg bw/day were administered daily by gavaging. Body weights and food and water intake were not significantly affected by the treatment and there were no treatment-related gross abnormalities. Upon microscopy, hepatocellular alterations in the periportal area of the liver (ground glass cytoplasm) were observed in the mid- and top-dose rats in both sexes. The incidence and severity of these alterations was treatment-related. Plasma gamma-glytamyltransferase activity and concentrations of the total protein, bilirubin, cholesterol and calcium were significantly increased in females of the top-dose group. A NOAEL of 3 mg/kg/ bw/day was determined for this study.

 

In the supporting study, the test compound was administered by gavaging to Sprague-Dawley rats at dose levels of 0, 10, 25, 50 or 100 mg/ kg bw/ day. The vehicle used for gavaging was corn oil. Mean body weights of control and treated males and females were similar throughout the study. No clinical signs of toxicity were noted that could be attributed to the test chemical. Gross examinations of tissues and organs at the terminal necropsy revealed no lesions that could be definitely attributed to the test chemical. However, treatment related adverse effects were observed during the serological and histopathological examinations, namely elevated levels of the liver enzymes SGOT, SGPT and GGTP and hepatocellular lesions consisting mainly of periportal degeneration and necrosis, respectively. Thus, also in the supporting study the results point into the direction of the liver as the target organ. A NOAEL could not be determined in this study.

Subchronic oral toxicity

A sub-chronic oral toxicity study (MR-350 (key)) is available that assesses the effects of 44PD after 92-94 days of exposure to rats via the diet. The tested concentrations in the diet are 0.002, 0.01 and 0.05%. The dietary concentration provided to the lowest dose group was raised to 0.1% after 65 days, and further to 0.2% after 90 days. The actual ingested doses depend on the mean food consumption and body weights of the animals. Male and female animals in the highest dose group received 60.7 mg/kg bw and 56.6 mg/kg bw at the beginning of the test, respectively; and the received dose had decreased to 26.1 mg/kg bw and 29.4 mg/kg bw by the end of the 13 week exposure period, respectively. Similarly, the doses for the rats in the mid-dose group at the beginning and end of the test decreased from 12.0 to 5.42 mg/kg bw for the males and from 12.4 to 6.38 for the female animals. The males in the lowest dose group received 2.48 mg/kg bw and the females received 2.56 mg/kg bw at the beginning of the test. At the end of the test, as the concentration of test substance in the diet was augmented to 0.2%, the actual ingested dose was 106.0 and 113.0 mg/kg bw for males and females, respectively.

The gross and microscopic evaluations and the clinical and haematological studies showed no treatment-related effects. However, the growth of the male rats of the high dose level was significantly lower than that of the controls at the conclusion of the experiment. Females at the high dose level showed a slight but statistically insignificant depression in weight. The animals in the mid dose level did not show significant differences in weight when compared to the controls. The mean body weight of the animals in the lowest dose group showed a marked descent after the dietary level was increased. From these data and taking into account the doses received at the end of the exposure period, a conservative NOAEL of 5,42 mg/kg bw for male and 6,38 mg/kg bw for female rats can be deduced.

Dose descriptor determination 

Bearing the precautionary principle in mind, the dose descriptor selected for the Chemical Safety Assessment is the NOAEL deduced from the key 28d study: 3 mg/kg bw.



Justification for classification or non-classification

The combined experimental data available for 44PD indicate that the liver is the main target organ for adverse effects upon oral administration of the test substance. As reported by Shellenberger, marked histopathological alterations were found in rats dosed with 50 and 100 mg/kg bw of test substance, including periportal degeneration and necrosis. Furthermore, the death of 2 animals of the highest dose groups that died on days 4 and 5 was most likely caused by liver failure. At dose levels below 50 mg/kg bw, the histopathological effects were less severe and fewer in number. As a consequence, 50 mg/kg bw was selected as the lowest dose that induces significant toxic effects in a 28 day oral exposure test in rats. Based on the guidance values (x3 in order to correct for 28d to 90d extrapolation) provided in table 3.9.3 in section 3.9 of Annex I to the CLP regulation, a classification as STOT RE Category 2 is warranted for this level of toxicity. The corresponding hazard statement is H373: Causes damage to organs (liver) through prolonged or repeated exposure. For the labelling the GHS08 pictogram (health hazard) and the signal word “Warning” are assigned to this category.

 

The classification according to Directive 67/548 (DSD) is “ Danger of serious damage to health by prolonged exposure” with R48 as the corresponding risk phrase.