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Description of key information

Based on the results of both studies a NOAEL of 50 mg/kg/d was taken forward to derive the DNEL.

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 02 August 1989 to 13 December 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Study conducted prior to the adoption of the most recent version of this guideline.
Deviations:
yes
Remarks:
The animals were treated for at least 120 days instead of 90; the neurobiological examination was not performed; it is not reported whether opthalmoscopic examination was conducted before the beginning of the treatment period.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, France
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation:
Mean initial bodyweight for males was 185 g
Mean initial bodyweight for females was 160 g
- Diet (e.g. ad libitum): a complete commercial diet ad libitum
- Water (e.g. ad libitum): tap-water through automatic waterers ad libitum
- Acclimation period: at least 1 week before beginning treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 60 +/- 10
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 02 August To: 13 December 1989
Route of administration:
oral: gavage
Vehicle:
other: 10% gum arabic solution
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The compound was prepared extemporaneously as a suspension in a 10% aqueous gum arabic solution

VEHICLE
- Justification for use and choice of vehicle (if other than water): no
- Concentration in vehicle: 10 %
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were carried out during the study to control the concentration of suspensions at approximately 3 months, 4 months and 4 months and 2 weeks after the beginning of the treatment period.
Duration of treatment / exposure:
At least 120 days (between 133 and 142)
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 5, 50 and 500 mg/kg bw/day
Basis:
other: nominal concentration
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
post-exposure period : no
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed at least twice daily (at least once on Saturdays, Sundays and public holidays) throughout the study for clinical signs (for which date of onset and progression were recorded) and for mortality.


BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed before the beginning of the treatment period, then weekly throughout the study.


FOOD CONSUMPTION : Yes
- Time schedule: individual food intake was measured weekly from which mean individual daily values were calculated on a weekly basis.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: day 126
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on days 128 and 129
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: 80
- Parameters checked in table [1] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 132 and 133
- Animals fasted: Yes
- How many animals: 80
- Parameters checked in table [2] were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: days 121 to 125
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- How many animals: 40
- Parameters checked in table [3] were examined.


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
Necropsy was performed from day 134 to day 143.

GROSS PATHOLOGY: Yes (see table 4 for tissues examined and table 5 for organ weights )
HISTOPATHOLOGY: Yes (see table 6)
Other examinations:
Not reported; no treatment-free period performed.
Statistics:
1. Parametric methods
Parametric, statistical tests are used when the theoretical distribution of the data is normal.
Step 1 consists of the Levene test for the equality of variances.
. If the Levene test cannot be performed or is not significant (equal variances), step 2 consists of testing for a treatment (group) effect using one-way analysis of variance (1-ANOVA).
- If 1-ANOVA is not significant, no further analysis is performed, the means being considered not different.
- If 1-ANOVA is significant (rejection of null hypothesis that population means are equal), step 3 consists of intergroup comparisons by the Student t test taking 1-ANOVA residual variance as a pooled estimate of the variability.

. If the Levene test is significant (unequal variances), step 2 consists of pairwise comparisons of variances (with the reference group) using the Snedecor F statistic.
- If a pairwise comparison is not significant (equal variances), step 3 consists of intergroup comparison by the classical Student t test.
- If a pairwise comparison is significant (unequal variances), step 3 consists of intergroup comparisons by the approximate Student t test using Welch's method.

To take into account multiple comparisons, p values are calculated according to Bonferroni's method (<= 6 comparisons) or Scheffé's method (> 6 comparisons).

2. Non-parametric methods
Non-parametric methods are routinely used when the theoretical distribution of the data is not normal.
Overall equality of the group means is tested by the Kruskall Wallis test. If the test is significant (unequal means), then intergroup comparisons are carried out pairwise by the same method. If the test is not significant, the means are considered to be not different.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Soiled urogenital area, blood on the muzzle and thinness were noted in one female given 50 mg/kg bw/day from day 100. Soiled urogenital area or blood on the muzzle was also noted, respectively, in one female or one male given 5 mg/kg bw/day. Hair loss was noted in one female given 50 mg/kg bw/day. These signs were not considered as related to the treatment with the compound.
One male given 5 mg/kg bw/day and one male given 500 mg/kg bw/day were found dead on days 61 and 60, respectively. Death was attributed to probable inhalation pneumonia following oesophageal occlusion as shown by the microscopic examination.


BODY WEIGHT AND WEIGHT GAIN
No treatment-related variations were noted in the body weight gain.

FOOD CONSUMPTION
A slight increase (sometimes statistically significant compared to controls) was noted in the animals given 500 mg/kg bw/day throughout the study.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related lesions were observed at the end of the treatment period.

HAEMATOLOGY
There were no relevant variations that can be related to the treatment with the test substance.

CLINICAL CHEMISTRY
An increase in total cholesterol level (+69% compared to the controls) was noted in males given 500 mg/kg bw/day. A slight decrease in creatininemia was noted in the test substance-treated groups without dose relationship. This change has no toxicological significance.

URINALYSIS
No treatment-related variations were noted.

ORGAN WEIGHTS
An increase in the absolute and relative kidney weight (approximately + 20%) was noted in the males given 500 mg/kg/day. As this was not associated with histological or biological modifications, this increase was considered to be of no toxicological significance.
A slight increase in the relative liver weight (with increase absolute values which however, were not statistically significant) was noted in the males and females given 500 mg/kg bw/day.

GROSS PATHOLOGY
The post-mortem examinations performed on all animals did no show any treatment-related lesions

HISTOPATHOLOGY: NON-NEOPLASTIC
A slight hypertrophy of centrilobular hepatocytes was noted in 2/10 males given 50 mg/kg/day and 10/10 males given 500 mg/kg bw/day. In 4/10 males of the high dose group, this was accompanied with a slight hypertrophy of the mediolobular hepatocytes. These changes were more or less associated with a decrease in the incidence of hepatocytes cytoplasmiques margination.

Histopathology of the decedent animals:
Moderate or marked acute pulmonary edema (congestion and alveolar edema) was seen in the two decedent animals, with perivascular edema and intra-alveolar hemorrhage in animal given 5 mg/kg bw/day and serosity in laryngeal and tracheal lumen in animal given 500 mg/kg/bw/day.
Moderate and marked distension of the oesophageal lumen by vegetal particles was noted and found also, mixed with serosity, in the tracheal and pharyngeal lumen in animal given 5 mg/kg bw/day.
Slight vacuolation of centrilobular hepatocytes was seen in animal given 5 mg/kg bw/day as well as moderate adrenocortical hyperplasia and various congestion changes in animal given 500 mg/kg/day. These changes are of little significance.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Slight liver hypertrophy in 2/20 animals without any other changes
Key result
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Liver: clinical chemistry; organ weights; histopathology Kidney: clinical chemistry; histopathology
Key result
Critical effects observed:
not specified

Table 7: Clinical chemistry - relevant/statistical changes

Exposure Group

Control

5 mg/kg/d

50 mg/kg/d

500 mg/kg/d

Males

Clinical Chemistry

Total

cholesterol nM

1.67 +/- 0.076

1.95 +/- 0.163

2.07 +/- 0.102

2.82 +/- 0.130*

* Statistically significant difference from the control group

Conclusions:
In this 4-month oral toxicity study, the NOAEL was found to be 50 mg/kg bw/day, based on a LOAEL estimated at 500 mg/kg bw/day (effects on liver in both sexes).
When Dibenzylbenzene, ar-methyl derivative (trade name: JARYLEC DBT) is administered at 50 mg/kg bw/day, slight hypertrophy of centrilobular hepatocytes was seen in few animals (2/20) without any other changes. Thus, this dose level was considered as the NOAEL.
Executive summary:

The aim of the present study was to assess the oral toxicity of Dibenzylbenzene, ar-methyl derivative (trade name: JARYLEC DBT) to Sprague Dawley rats. The compound was administered once a day by gavage for at least 120 days at the dose levels of 0, 5, 50 and 500 mg/kg bw/day. Each group was composed of 10 males and 10 females. The control group received the vehicle only (l0 % gum arabic solution).

No treatment-related mortality or clinical signs were noted and the growth rate of animals was unmodified.

Ophthalmoscopic and hematological examinations, urinalysis and macroscopic examinations did not show any treatment-related lesions.

At 500 mg/kg bw/day, the compound induced changes suggesting an enzyme induction: hypertrophy of centrilobular (and sometimes mediolobular) hepatocytes in most animals associated with a slight increase in cholesterol levels in males, a slight increase in food intake and in the relative liver weight in both sexes.

A slight increase in kidney weight was noted in high-dose males, but it was considered without toxicological relevance since it was not associated with histological or biological modifications.

At 50 mg/kg bw/day, only two males showed a slight hypertrophy of centrilobular hepatocytes without any other changes; Thus, this dose level was considered as a non-toxic dose. Consequently, under the experimental conditions of this 4-month oral toxicity study, the NOAEL was found to be 50 mg/kg bw/day, based on a LOAEL estimated at 500 mg/kg bw/day (effects on liver in both sexes).

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

Additional information

The toxicity of Dibenzylbenzene, ar-methyl derivative after repeated exposure was evaluated in 2 independent studies in rats with daily oral gavage administration over 3 or 4 months, respectively.

In the 3 months study doses of 10, 100 or 500 mg/kg/d were applied by oral gavage. The test substance caused dose-related increases in liver and kidney weights in both males and females at doses of 100 and 500 mg/kg bw/d. All other effects (effects on haematology, clinical chemistry, histopathology) are considered coincidental and/or without biological importance as they were almost completely reversible after a recovery period of 4 weeks. Based on the available data the dose of 10 mg/kg/d was considered as NO(A)EL under the conditions of this study.

In the 2nd study rats received the test substance at dose levels of 0, 5, 50 and 500 mg/kg/d via oral gavage. No treatment-related mortality or clinical signs were noted and the growth was not affected. At 500 mg/kg bw/day, the compound induced changes suggesting an enzyme induction: hypertrophy of centrilobular (and sometimes mediolobular) hepatocytes in most animals associated with a slight increase in cholesterol levels in males, a slight increase in food intake and in the relative liver weight in both sexes.

A slight increase in kidney weight was noted in high-dose males, but it was considered without toxicological relevance since it was not associated with histological or biological modifications.

At 50 mg/kg bw/day, only two males showed a slight hypertrophy of centrilobular hepatocytes without any other changes. Thus, this dose level was considered as non-toxic dose and hence under the conditions of this 4-month oral toxicity study, the NOAEL was found to be 50 mg/kg/d.

Based on the results of both studies a NOAEL of 50 mg/kg/d was taken forward to derive the DNEL.

Justification for classification or non-classification

No classification for repeated dose effects or target organ toxicity is indicated according to the general classification and labeling requirements for dangerous substances and preparations (67/544 EEC) or the classification, labeling and packaging (CLP) regulation (EC 1272/2008).