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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:
From 26 July 1989 to 13 decembre 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliance and Guideline study (OECD 408, May 12, 1981) with few deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
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:
Animals treated for at least 120 days instead of 90; neurobiological examination was not performed; it is not reported whether opthalmoscopic examination was conducted at study start.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): JARYLEC BT

Test animals

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 (160 - 207 g)
Mean initial bodyweight for females was 160 g (140 - 175)
- Fasting period before study: not reported
- Housing: individually; space allocated: 345 square cm x 17 cm
- Diet: a complete commercial diet ad libitum
- Water: 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: 2 August To: 13 December 1989

Administration / exposure

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% aqueous gum arabic solution
- 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 start of treatment.
Duration of treatment / exposure:
at least 120 days
Frequency of treatment:
7 days/week.
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure period: none
- Dose selection rationale: no
Positive control:
No

Examinations

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: Animais were weighed before treatment started, then weekly throughout the study.


FOOD CONSUMPTION :
- 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: on Day 126
- Dose groups that were examined: on 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 [No.?] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on days 132 and 133
- Animals fasted: Yes
- How many animals: 80
- Parameters checked in table [No.?] were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: on days 121 to 123
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- How many animals: 40 (5/sex/dose)
- Parameters checked in table [No.?] were examined.


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Necropsy was performed from day 134 to day 143.
GROSS PATHOLOGY: Yes (see table 4 for tisues examined and table 5 for organ weights)
HISTOPATHOLOGY: Yes (see table 6)
Other examinations:
Not reported
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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
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 specified
Details on results:
CLINICAL SIGNS AND MORTALITY
Hair loss was noted in 3 males and 7 females receiving the high dose (500 mg/kg/d). This sign mainly affected the abdomen, flanks and thighs. Four females of the same group presented soiled urogenital area.
These changes could be attributed to treatment whereas thinness, observed in two high-dose females could be considered incidental since one of the two animais was found dead with intercurrent pathology a few days Iater.
Blood on the muzzle, noted in a few treated animals, was not attributed to treatment.
One female treated at 500 mg/kg bw/d died on Day 46. This death may have been due to gastric inflammation. As the stomach is not a target organ for Jarylec BT, the death of this animal cannot be directly attributed to the product, but rather to a spontaneous pathology of this animal.

BODY WEIGHT AND WEIGHT GAIN
The bodyweight gain of high dosed (500 mg/kg/d) animals was decreased throughout the study, although this change was only statistically significant only in females. On Day 120, the bodyweight of males was 10 % lower than that of controls while that of females was 15 % Iower.
The other variations were not attributed to treatment.

FOOD CONSUMPTION
A slight increase in food intake was noted in high-dose (500 mg/kg/d) males throughout the fourth month of the study and in high-dose females throughout months 3 and 4 of the study.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related lesions were observed.

HAEMATOLOGY
The following variations were attributed to treatment:
. A tendency towards anemia in high-dose (500 mg/kg/d) animals characterized by:
- a slight decrease in hemoglobin levels (7 to 9%),
- a slight decrease in the erythrocyte count (approximately 10 %) and packed cell volume (PCV) (11 to 15 %),
- an increase in the mean corpuscular hemoglobin concentration (MCHC) in both sexes,
- a decrease in the mean corpuscular volume (MCV) in females only.
. An increase in MCHC was also observed in mid-dose (50 mg/kg) females.
. A slight increase in the leukocyte count in high-dose (500 mg/kg/d) males (+ 32 %) due to an increase in neutrophil and lymphocyte counts.

The other variations were not attributed to treatment. Examination of bone marrow smears was not considered necessary.

CLINICAL CHEMISTRY
The following variations were attributed to treatment:
. Slight increase in creatinine levels (+ 16 %) and albumin levels (+ 10 %) in high-dose (500 mg/kg/d) males.

URINALYSIS
The following variations were attributed to treatment:
. Non statistically significant increase in specific gravity in high-dose (500 mg/kg/d) animals.
. Increased chloride (+ 78 %) excretion in high-dose (500 mg/kg/d) females.
. Increased percentage of animals excreting small amounts of bilirubin in urine in the high-dose group (500 mg/kg/d) compared with controls:
- Control group: M (1/5) - F (0/5)
- High-dose group: M (3/5) - F (4/5)

The other variations were not attributed to treatment.

ORGAN WEIGHTS
The following variations were attributed to treatment:
. Increased absolute liver weight (+ 23 %) in high-dose (500 mg/kg/d) males and females with increased relative weight (30%).
The other variations were not attributed to treatment. The slight increase in the kidney's relative weight in high-dose (500 mg/kg/d) animals and the increase in the relative uterus weight in high-dose females, were atttributed to the lower bodyweight of these animals compared to controls.

GROSS PATHOLOGY
No treatment-related variations were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC (attached illustration)
Treatment-related findings:
. Liver:
- Major effect observed was a slight hypertrophy of centrilobular hepatocytes in one male and one female given 50 mg/kg/d and in most animais given 500 mg/kg/d. A few chronic inflammatory cell foci and slight to moderate periportal hepatocyte marginations were also reported.

. Kidney:
- increased intensity of brown pigmentation of proximal tubular cells, only in group 3 males (500 mg/kg/d).
It is associated with a greater number of males of this group exhibiting hyalin droplets inside these cells (such hyalin droplets were observed only in case of moderate brown pigmentation).

Based on the changes observed in the female (500 mg/kg/d) found dead on Day 46 during the study, it was considered that the death was due to health deterioration following gastric inflammation and finally, acute pumonary oedema.

Other findings were physiological or belong to the spontaneous pathology of the species.

Effect levels

open allclose all
Key result
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 biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 7 Mean Body Weights (g) – Most relevant changes

Exposure Group

Control

5 mg/kg/d

50 mg/kg/d

500 mg/kg/d

Males

Day 1

187 +/- 2.5

185 +/- 2.1

193 +/- 1.9

185 +/- 3.6

Day 120

567 +/- 17.9

609 +/- 20.3

599 +/- 10.6

511 +/- 17.4

Body weight change

+ 380

+ 424

+ 406

+ 326

Females

Day 1

166 +/- 1.9

161 +/- 2.8

158 +/- 2.6

166 +/- 1.8

Day 120

349 +/- 8.0

337 +/- 8.6

355 +/- 12.4

296 +/- 10.6*

Body weight change

+ 183

+ 176

+ 197

+ 130*

*Statistically significant difference from the control group

Table 8 Haematology and clinical chemistry – Statistical changes

Exposure Group

Control

5 mg/kg/d

50 mg/kg/d

500 mg/kg/d

Males

Haematology

RBC (10E3/mm3)

9276 +/- 125

9130 +/- 178

8721 +/- 112

8323 +/- 180*

Hb (g/L)

160 +/- 2

156 +/- 3

156 +/- 2

149 +/- 2*

PCV (%)

47 +/- 0.7

46 +/- 0.9

46 +/- 0.6

42 +/- 1.1*

MCHC (%)

33.9 +/- 0.2

33.6 +/- 0.2

33.8 +/- 0.3

35.5 +/- 0.5

Leukocytes (10E3/mm3)

8.9 +/- 0.6

9.8 +/- 0.7

8.2 +/- 0.4

11.9 +/- 0.6*

Clinical Chemistry

Albumin

37.2 +/- 0.9

39.4 +/- 0.6

39.1 +/- 0.4

40.9 +/- 0.7*

Females

Haematology

RBC (10E3/mm3)

8558 +/- 85

8659 +/- 119

8533 +/- 135

7081 +/- 159*

Hb (g/L)

155 +/- 2

159 +/- 2

159 +/- 2

142 +/- 3*

PCV (%)

46 +/- 0.4

46 +/- 0.6

45 +/- 0.6

39 +/- 0.9*

MCV (µmE3)

53.5 +/- 0.6

53.4 +/- 0.5

52.8 +/- 0.6

50.5 +/- 0.8*

MCHC (%)

34.0 +/- 0.2

34.4 +/- 0.2

35.3 +/- 0.2

36.2 +/- 0.3*

*Statistically significant difference from the control group

Table 9 Urinalysis – Statistical changes

Exposure Group

Control

5 mg/kg/d

50 mg/kg/d

500 mg/kg/d

Males

Specific gravity

1.018 +/- 0.001

1.018 +/- 0.002

1.017 +/- 0.002

1.027 +/- 0.003

Females

Specific gravity

1.017 +/- 0.003

1.020 +/- 0.004

1.020 +/- 0.003

1.024 +/- 0.003

Chlorides (mM)

38 +/- 5.2

69 +/- 7.5

65 +/- 7.4

81 +/- 10.4*

*Statistically significant difference from the control group

Table 10 Organ Weights (g) – Most relevant changes

Exposure Group

Control

43 mg/kg/d

84 mg/kg/d

170 mg/kg/d

Males

Absolute organ Weight (g)

Liver

18.2 +/- 0.9

19.4 +/- 1.0

17.8 +/- 0.7

22.4 +/- 0.9*

Relative organ Weight (g/kg) to bodyweight

Liver

31.7 +/- 0.9

31.7 +/- 0.8

30.3 +/- 0.6

42.9 +/- 0.7*

Kidney

6.3 +/- 0.2

6.5 +/- 0.2

6.5 +/- 0.1

7.4 +/- 0.1*

Females

Absolute organ Weight (g)

Liver

10.2 +/- 0.2

9.33 +/- 0.3

10.0 +/- 0.4

12.5 +/- 0.6*

Relative organ Weight (g/kg) to bodyweight

Liver

30.1 +/- 1.0

28.5 +/- 0.6

29.0 +/- 0.7

42.6 +/- 2.1

Kidney

6.5 +/- 0.2

6.3 +/- 0.1

6.5 +/- 0.2

7.6 +/- 0.3*

*Statistically significant difference from the control group

Applicant's summary and conclusion

Conclusions:
In this 4-month oral toxicity study in rats, the NOAEL was found to be 50 mg/kg bw/d, based on a LOAEL estimated at 500 mg/kg bw/d (effects on liver in both sexes and kidney only in males).
When administered at 50 mg/kg/d, Jarylec BT induced slight hypertrophy of centrilobular hepatocytes in a few animals only (2/20) without any other changes. As a result, this dose level was considered as the NOAEL.
Executive summary:

Benzyl toluenes was administered by gavage as suspension in 10% arabic gum at the dose-levels of 0, 5, 50 and 500 mg/kg bw to four groups of 20 sprague-dawley rats (10 rats/sex/group). The clinical signs were observed twice a day, the body-weight and the food consumption were recorded once a week. For all animals, ophthalmology, clinical pathology, hematology investigations and urine analysis were performed after at least 120 days of treatment. After at least 120 days of treatment, the rats were sacrificed and a full macroscopic examination was performed, selected organs weighted, and certain organs submitted to histological examination.

No behavioral changes were noted. Ophthalmoscopic examinations did not show any treatment-related lesions. The growth rate of high-dose (500 mg/kg bw) animals was slightly lower than that of controls throughout the study. This difference was not statistically significant. Food consumption was unaffected by the treatment. Hematological examinations showed tendency towards anemia in female rats treated at 500 mg/kg bw. No effect was observed in males, and in females of the 5 and 50 mg/kg bw groups. Blood biochemical examinations and urine analysis did not show any significant modifications. The study of organ weights showed an increase in the relative liver weight in high-dose males (+35%) and female (+41%), an increase in the relative kidney weight in high-dose males (+17%). Light microscopic examination revealed that the administration of benzyl toluenes induced in the 500 mg/kg bw group an increased accumulation of hyaline globules in the renal proximal tubular cells in male animals, and a minimal hypertrophy of hepatocytes in male and female rats.

On the basis of these results, the no-effect level for benzyl toluenes administrated to rat during a 120 day period can be estimated at 50 mg/kg bw.