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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015 - 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: bulk
Details on test material:
- Test item name: 2-Acetone, condensation product with phenol
- Appearance: brownish solid
- Batch no.: 2015-05-30
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2015-05-30
- Expiration date of the lot/batch: 2016-05-20

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: analytically confirmed
- Solubility and stability of the test substance in the solvent/vehicle: analytically confirmed
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none
Stability for at least 5 hours at room temperature and for at least 8 days in the refrigerator in the dark is confirmed over the concentration range 1 to 200 mg/mL, Test Facility Study No. 510051.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance 2-Acetone, condensation product with phenol was filled at 130 °C into plastic covered Duran glass bottles. During the cooling down a vacuum was build which tightens the cap. Therefore, the bottles need to be heated up to 60°C in a cabinet heater before the cap could be loosed. Decanting was not possible at this temperature. Therefore, the bottles need to be heated up to 100-120 °C in a cabinet heating with loosed cap. Then decanting was possible in a fume cupboard with appropriate heat protection gloves.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks
- Fasting period before study: no
- Housing: 5 animals per sex in Macrolon cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days before start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 °C
- Humidity (%): 40-70%
- Air changes (per hr): at least 10 air changes/hours
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From 2015-09-30 to 2016-02-26 (including dose range finding study)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: polyethylene glycol 400, specific gravity 1.125
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch and on information from the sponsor.
- Amount of vehicle (if gavage): 5 mL/kg bw

METHOD of FORMULATION:
Formulations (w/w) were prepared within 8 days prior to dosing and were homogenized to visually acceptable levels. The formulations were heated on the day of formulation to a maximum of 80±5°C for at least 15 minutes to obtain visual homogeneity. Formulations were released for dosing when they had obtained a temperature of 40°C or lower. Adjustment was made for specific gravity of the vehicle. No correction was made for purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability measurements were conducted as part of the method development and validation study (ABL Project 15273). The stability of the test item in the vehicle was assured prior to the start of the main study.
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in Weeks 1, 7 and 13).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

RESULTS:
No test item was detected in the control group formulations.
The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).
The formulations of Group 2 and Group 4 were homogeneous in all groups analysed (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
at least 90 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10/sex/dose
control female 50 was replaced at day 8 due to an accidental death on day 7 and treated one week less with the vehicle
Control animals:
yes, concurrent vehicle
no
Details on study design:
- Dose selection rationale:
Dose levels are based on results of a 28-day oral range finding study with 2-Acetone, condensation product with phenol by daily gavage in the rat (Test Facility Study No. 509883).
Positive control:
not adequate

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena (collected under Test Facility Study No. 510847 for logistic reasons and reported under Test Facility Study No. 509882). The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption: weekly

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: Yes / No / Not specified

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Dose groups that were examined: all animals at pretest; groups 1 and 4 animals at week 13.
Since no treatment-related ophthalmologic findings were noted in Week 13, the eyes of the rats of Groups 2 and 3 were not examined.

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 12-13
- Dose groups that were examined: forst 5 animals/sex/group
- Battery of functions tested: hearing activity / pupillary reflex / satic righting reflex / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test (Ref. 5) was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs were predominantly noted for the animals at 250 mg/kg bw/day and consisted of rales, hunched posture, piloerection and/or labored respiration in three males and three females. At low incidence hunched posture, piloerection and/or rales were noted at 50 mg/kg bw/day in one male and one female. These clinical signs were not noted for the animals at 10 mg/kg bw/day.
Description (incidence):
There was one unscheduled death. Male no. 31 (250 mg/kg bw/day) was sacrificed moribund condition at Day 77. During two weeks prior to death this animal showed, hunched posture, laboured respiration and rales. In addition swelling of the abdomen and lean appearance were noted in this animal on the last two days prior to death.
At macroscopic examination the male was emaciated, showed a gastro-intestinal tract distended with gas, tan discoloration of the thymus and a reduced size of the thymus, mesenteric lymph node and seminal vesicles. Relevant microscopic findings consisted of atrophy (up to moderate degree) of thymus, mesenteric lymph node and seminal vesicles correlating with the reduced size of these organs. Since this unscheduled death was incidental and histopathological findings were unique in nature and/or severity in this study, a relation to treatment was doubted but could not be excluded.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain was noted in males at 250 mg/kg bw/day, achieving a statistically significant difference in from week 5 onwards. Statistical significance was also achieved for the lower body weights in males from week 9 onwards.

Body weights and body weight gain of female animals remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight remained similar to the control level over the study period.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in haematological parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical biochemistry parameters distinguished treated from control animals:
- Higher alanine aminotransferase in males at 250 mg/kg bw/day
- Higher alkaline phosphatase in females at 250 mg/kg bw/day
- Lower albumin in females at 50 and 250 mg/kg bw/day
- Higher urea in females at 250 mg/kg bw/day (non-statistically significant at 50 mg/kg bw/day)
- Lower cholesterol in both sexes at 50 and 250 mg/kg bw/day (not statistically significant in males at 50 mg/kg bw/day)
- Higher inorganic phosphates in both sexes 250 mg/kg bw/day

Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Grip strength of the hind limps was lower in males and females at 250 mg/kg bw/day. Grip strength of the fore limp was similar between control and high dose animals.

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.

Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant test item-related higher liver weights (relative to body weights) were noted in males from 50 mg/kg bw/day onwards (up to 5% absolute and 20% relative) and in females at 250 mg/kg bw/day (8% absolute and 13% relative).

Some organ weight differences in males were statistically significant when compared to the control group but were considered to be the result of a test item-related effect on final body weight.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic findings which were considered to be related to the treatment with the test item were noted at 250 mg/kg bw/day in the liver of both sexes. These findings consisted of hepatocellular hypertrophy (minimal) to 3/9 in males and 6/10 in females.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Some slight clinical biochemistry changes were found in the high dose group (such as higher alanine aminotransferase, alkaline phosphatase, urea and/or inorganic phosphates and lower albumin and cholesterol levels).

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
NOAEL subchronic oral toxicity study in rats: 50 mg/kg bw/day
Executive summary:

A subchronic oral gavage toxicity study in rats following OECD 408 was performed with the substance. Based on toxicity observed in a 28-day range finding study the dose levels were selected with 0, 10, 50 and 250 mg/kg bw/day. Formulation analyses confirmed that formulations of test item in polyethylene glycol 400 were prepared accurately and homogenously.

There was one male at 250 mg/kg bw/day sacrificed due to moribund condition at Day 77. During two weeks prior to death this animal showed clinical signs as, hunched posture, laboured respiration and rales. At macroscopic examination the male was emaciated, showed a gastro-intestinal tract distended with gas. Relevant microscopic findings consisted of atrophy (up to moderate degree) of thymus, mesenteric lymph node and seminal vesicles correlating with the reduced size of these organs. Although these findings were considered incidental, a relation to treatment with test item cannot be excluded based on similar clinical signs seen in another animal of this dose group. Clinical signs in the surviving animals were noted in 3/9 males and 3/10 females at 250 mg/kg bw as rales and hunched posture. In addition, reduced body weight (-11% in males) and body weight gain (-20% in males and -10% in females) was seen at the high dose in combination with a and reduced grip strength of the hind limbs. The latter can be considered as non-specific effect of toxicity within historical control data. At histopathological examination test item-related microscopic findings were present at 250 mg/kg bw/day in the liver of both sexes (3 males and 6 females) and consisted of hepatocellular hypertrophy at a minimal degree. This was correlated in both sexes with higher liver weights (males + 20% and females + 13% relative to body weight). Some slight clinical biochemistry changes were found in this study (such as higher alanine aminotransferase, alkaline phosphatase, urea and/or inorganic phosphates and lower albumin and cholesterol levels).

At 50 mg/kg bw/day clinical signs were seen in one male (rales, 1 day of treatment) and one female (temporary hunched posture and piloerection) only. A slight increase in relative liver weight was recorded in males. Based on the low severity and absence of other test item-related and/or histopathological effects, these findings were considered to be not adverse.

No compound related adverse findings were observed at 10 mg/kg bw/day.

Based on the results in this subchronic toxicity study, the No Observed Adverse Effect Level (NOAEL) for 2-Acetone, condensation product with phenol was determined at 50 mg/kg bw/day.