Benzyltoluene

Endpoint summary

Description of key information

Two key studies are available for oral repeated dose toxicity of benzyl toluene.

 

The first one (Verschuere, 1992) is a 4-month study that was conducted on rats using benzyl toluene of very high purity. The study was conducted according to OECD 408 guideline, with minor deviations (120 days exposure duration, ophthalmologic examination and neurobiological examination not performed) and was GLP. Benzyl toluene 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 toluene 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 toluene administrated to rat during a 120 day period can be estimated at 50 mg/kg bw.

 

The other key study (Korn, 1990) had similar conclusion with same NOAEL and LOAEL.

The potential toxicity of benzyl toluene was evaluated following repeated oral administration for 13 weeks, according to OECD Guideline 408. Benzyl toluene was administered once daily by gavage to Sprague-Dawley rats (10 Males and 10 Females), at the dose-levels of 0, 5, 50 and 500 mg/kg/d during 90 days. Body weights were recorded pre-test, weekly and at death or prior necropsy. Animals were observed daily for toxicity and pharmacological effects, and twice daily for morbidity and mortality. Food consumption was calculated weekly. A neurobehavioral examination was performed. Whole blood, serum and plasma were sampled just prior necropsy and analyzed for related hematological and clinical chemistry parameters. All animals were examined for gross pathology, and organs were weighted and submitted to histopathology.

The No Observed Adverse Effect Level (NOAEL) for repeated dose toxicity was considered to be 50 mg/kg bw/d based on effects on liver weights and bilirubin level. All other findings in hematology, clinical chemistry and histopathology are considered to be coincidental and/or of no biological importance as they were completely reversible after a recovery period of 4 weeks.

 

There is also a 2 weeks oral toxicity study performed as preliminary study to the OECD 421 (CRL, 2021): in this study, the toxicity of the test item, Benzyltoluene, was evaluated after daily oral administration (gavage) to Sprague-Dawley rats at the dose levels of 100, 300, 600 and 800 mg/kg bw/day for 2 weeks.
Based on the results of this study, the doses of 600 and 800 mg/kg bw/day were considered to exceed the Maximum Tolerated Dose (MTD) in males and females.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study 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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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

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.

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

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.

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.

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

histopathology: non-neoplastic

organ weights and organ / body weight ratios

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

histopathology: non-neoplastic

organ weights and organ / body weight ratios

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

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

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

March to July 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented study performed according to OECD test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted May 12, 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH
- Age at study initiation: about 6 weeks
- Weight at study initiation: males: 111 to 143 g; females: 100 to 133 g
- Fasting period before study: no data
- Housing: single in Makrolon type II cages
- Diet: Ssniff R diet in pellet form produced by Ssniff Spezialdiäten Gmbh, Soest, Germany (ad libitum)
- Water: tap water (ad libitum)
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 +/- 1.5 °C
- Humidity (%): 60 +/- 10%
- Air changes: 16 per hr
- Photoperiod: 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: March 21, 1990 To: June 27, 1990 or July 26, 1990 (recovery groups)

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % sodium carboxy methylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was diluted with vehicle to give final concentrations of 0.05, 0.5 and 5.0 %. Suspensions were homogenized by means of an Ultra turrax for 1 min with increasing numbers of revolutions and kept on a magnetic stirrer throughout administration. Samples were freshly prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 0.05, 0.5 and 5.0 %
- Amount of vehicle (if gavage): 10 ml/kg bw
- Lot/batch no. (if required): no data
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of dosing solutions on treatment days 1, 43 and 92. Additionally a 5 % test article suspension was analysed 2, 6 and 24 hours after preparation to determine stability.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

once daily

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, additional 10 animals per sex in control and high dose groups for recovery period

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose selection according to results of a range finding study
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 4 weeks

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily obervations on sensory, motor behavior, coat, body orifices, urine, feces and general health status; mortality checks twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: at weekly intervals

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at weeks 0, 7, 14 and 17
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during weeks 13 and 18 (recovery group)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals per sex per group
- Parameters were examined: Erythrocytes (RBC), Leucocytes (WBC), Thrombocytes, Hemoglobin, Hematocrit, MCV, MCH and MCHC, differential blood count, Reticulocytes, Inclusion bodies and Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at weeks 14 and 18 (recovery group)
- Animals fasted: No data
- How many animals: 10 animals per sex per group
- Parameters were examined:
Substrates: Bilirubin, Creatinine, Glucose, Urea Nitrogen (BUN) and Uric Acid
Lipids: Triglycerides, Cholesterol
Proteins: Protein (toal), Albumin
Electrolytes: Calcium, Chlorine, Inorg. Phosphorus, Iron, Potassium, Sodium, Na/K ratio by statistical evaluation
Enzymes: Glutamate Dehydrogenase (GLDH), Alanine AMinotransferase (ALT), Aspartate Aminotransferase (AST), Creatinine Kinase (CK), Alkaline Phosphatase and AST/ALT ration by statistical evaluation

URINALYSIS: Yes
- Time schedule for collection of urine: at study initiation and at weeks 13 and 17
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No, before urine collection 5 animals/sex of all groups received an intragastric administration of 20 mg/kg water and were housed individually in metabolic cages over a period of 18 hours
- Parameters were examined: Color, Protein, pH value, Glucose, Bilirubin, Urobilinogen, Blood, Nitrite, Ketones, Sediment and specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to study initiation and after 6 and 13 weeks and at the end of the recovery period
- Dose groups that were examined: all groups
- Battery of functions tested: awareness, emotion, coordination, reflexes and autonomic functions

OTHER: hearing

Sacrifice and pathology:

GROSS PATHOLOGY: Yes ; examination on all animals; inspection of the cranial, thoracic, abdominal and pelvic cavities; determination of organ weights (see table 1)
HISTOPATHOLOGY: Yes ; examination on 10 male and 10 female animals each of control and high dose group; tissues examined (see table 2)

Statistics:

Statistical analysis were performed separately on data from male and female animals. One- or two-factorial analysis of variance was conducted on weight changes and food consumption. Group means were compared by the "Scheffé" test. The ratio (weight changes per week / food consumption per week x 100) was calculated (food conversion). Organ weights were evluated by analysis of covariance, animal weight being the independent variable and organ weight the dependent variable. Mean values were compared by the "Scheffé" method for the analysis of co-variance. Values from clinical chemistry and hematology were analysed by analysis of variance with subsequent Scheffé test for analysis of variance. Significance levels were indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

References:
- Winer, B. J. (1971) Statistical Principles in Experimental design; McGraw Hill, Kogakusha Ltd.
- Snedecor, G. W. and Cochran, W. G. (1969) Statistical methods, Sixth edition, IOWA State University Press

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):

no effects observed

Food efficiency:

no effects observed

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:

no effects observed

Behaviour (functional findings):

no effects observed

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:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment related clinical effects were observed, except of salivation in individual animals of high dose group immediately after treatment.
Two animals, i. e. one male of the low dose group and one female of the high dose group, were killed in extremis in week 8 and 2, respectively, following a deterioration in its condition as a result of faulty administration.

BODY WEIGHT AND WEIGHT GAIN
A slight statistically not significant declined weight gain rate were observed in males of the high dose group during the entire administration period. (see table 3)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No treatment related effects observed.

FOOD EFFICIENCY
No treatment related effects observed.

OPHTHALMOSCOPIC EXAMINATION
No treatment related effects observed.

HAEMATOLOGY
Slight reductions within normal ranges (statistically significant) were determined at the end of the administration period in erythrocyte and hematocrit values of males of the high dose group and in hemoglobin and hematocrit values of females of the mid and high dose groups. Prothrombin time values were slightly reduced below normal in the male animals of the high dose group. (see table 4)

CLINICAL CHEMISTRY
Biochemical analysis showed a dose-related reduction in Triglyceride values in males of the mid dose group and in both sexes of the high dose group. Group mean values differt significantly against control values and decreased towards the lower limit of the normal range in the high dose group.
Iron values were subnormal on high dose males and a dose-related trend declined bilirubin values was found in mid dose females and in both sexes of the high dose group. Slight reductions within the limit of normal range were found in sodium (high dose females), potassium (high dose males), calcium (mid and high dose males) and chloride (high dose males and females).
Na/K-ration was slightly in creased in high dose males.
Significant changes in cholesterol and inorganic phosphorus are considred to be due to statistical coincidence. GLDH results were heterogenous (high standard deviations) in all male groups and in mid dose females, possibly due to slight hemolysis of blood samples. (see table 5)

URINALYSIS
No treatment-related differences were observed.

NEUROBEHAVIOUR
No treatment-related effects were observed.

ORGAN WEIGHTS
In all high dose animals liver and kidney weights were significantly increased. In high dose females also an trend towards increased adrenal weights was found. (see table 6)

GROSS PATHOLOGY
No gross pathological organ alterations were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
Hepatocellular hypertrophy in 8 of 10 male and 8 of 10 female animals of the high dose group. In mid dose animals only 1 of 10 male and 2 of 10 female animals showed this finding. Low dose animals were unaffected.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No treatment-related effects were observed.

OTHER FINDINGS
Auditory examinations revealed no findings related to the administration of the test substance.

RESULTS OF THE RECOVERY GROUP
Body weight gain rates did not differ distinctly between control and high dose group recovery males at the end of the recovery period. However, a complete compensation of previous losses was not attained. No persisting significant differences were determined in hematology parameters. Newly determined statistically significantly reduced values in hemoglobin (high dose males) and MCHC (high dose males and females) are considered coincidental, since not corresponding changes were found at the end of the treatment. Increased prothrombin values in male animals were observed. These may be considered as compensatory effect to the reduction, as it was observed at treatment termination. Similar signs of normalization were determined for most parameters of clinical chemistry, except of a furtheron slight decline of bilirubin values in high dose females. Significant changes in AST/ALT ratio, GLDH, CK, uric acid, potassium, sodium and calcium did not correspond to findings of the treatment period and are therefore without biological importance.
Organ weight analysis still dislosed a slight increase of liver weights in high dose females, but the difference to control livers was less severe than at the end of the treatment period. Liver weights of male animals, kidney weights of both sexes and adrenal weights of females had returned to normal by end of the recovery week.
A slight decline of prostate weights did not correspond to results of the treatment period and is thus considered coincidental.
The hepatocellular hypertrophy underwent almost complete recovery with only 2 of 10 male and 4 of 10 female animals showing this finding.

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

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

Table 3: Body weight gain of males (mean values and standard deviations)
Group		Control	5 mg/kg	50 mg/kg	500 mg/kg
Weeks 0-6	absolute (g)	251.9 ± 24.7	247.9 ± 31.6	245.3 ± 16.0	227.6 ± 38.5
	relative (%)	100.0	98.4	97.4	90.4
Weeks 0-13	absolute (g)	361.0 ± 39.4	352.0 ± 43.9	342.9 ± 23.2	319.8 ± 45.3
	relative (%)	100.0	97.5	95.0	88.6
Weeks 6-13	absolute (g)	109.1 ± 19.2	98.87 ± 20.7	97.6 ± 12.3	92.2 ± 31.3
	relative (%)	100.0	90.5	89.5	84.5
Weeks 13-17	absolute (g)	33.1 ± 11.9			33.5 ± 16.2
(recovery groups)	relative (%)	100.0			101.2
Weeks 13-15	absolute (g)	17.0 ± 8.2			14.3 ± 13.8
(recovery groups)	relative (%)	100.0			84.1
Weeks 15-17	absolute (g)	16.1 ± 6.9			19.2 ± 5.1
(recovery groups)	relative (%)	100.0			119.3
Table 4: Haematology findings (mean values and standard deviations)
Group		Control	5 mg/kg	50 mg/kg	500 mg/kg	control recovery	500 mg/kg recovery	range of normal values - rat (Wistar)
Erythrocytes (10^12/l)	males	9.605 ± 0.389	9.449± 0.398	9.256 ± 0.321	8.862 ± 0.358**	9.480 ± 0.399	9.335± 0.486	5 - 9
	females	9.016 ± 0.332	8.803 ± 0.378	8.609 ± 0.369	8.611 ± 0.320	8.596 ± 0.327	8.707± 0.345	5 - 9
Hematocrit	males	0.5090 ± 0.0171	0.5040 ± 0.0131	0.4905 ± 0.0188	0.4781 ± 0.0267*	0.5062 ± 0.0138	0.5000 ± 0.0194	0.3 - 0.6
	females	0.5012 ± 0.0174	0.4801 ± 0.0175	0.4733 ± 0.0229*	0.4727 ± 0.0170*	0.4746 ± 0.0183	0.4848 ± 0.0184	0.3 - 0.6
Hemoglobin (mmol/l)	males	10.18 ± 0.27	10.26 ± 0.30	9.83 ± 0.39	9.66 ± 0.64	10.40 ± 0.30	10.04 ± 0.37*	7.0 - 10.0
	females	10.33 ± 0.29	9.88 ± 0.34	9.73 ± 0.42**	9.75 ± 0.33**	9.88 ± 0.42	9.82 ± 0.39	7.0 - 10.0
prothrombin time (%)	males	102.8 ± 4.6	105.3 ± 10.8	107.9 ± 5.5	88.2 ± 12.7*	102.2 ± 6.4	113.0 ± 10.6*	90 - 125
	females	121.0 ± 4.8	122.1 ± 6.2	120.0 ± 5.8	125.1 ± 4.1	128.8 ± 7.0	129.1 ± 7.3	90 - 125
*p < 0.05
** p < 0.1
***p < 0.001
Table 5: Clinical Chemistry (mean values and standard deviations)
Group		Control	5 mg/kg	50 mg/kg	500 mg/kg	control recovery	500 mg/kg recovery	range of normal values - rat (Wistar)
Triglyceride (mmol/l)	males	2.664 ± 0.700	2.327 ± 0.813	1.528 ± 0.511**	0.721 ± 0.316***	2.455 ± 0.499	2.139 ± 0.568	0.6 - 2.3
	females	1.809 ± 0.896	1.490 ± 0.438	1.115 ± 0.459	0.601 ± 0.269***	2.132 ± 1.354	1.234 ± 0.387	0.6 - 2.3
Iron (µmol/l)	males	33.46 ± 4.16	37.80 ± 6.84	33.44 ± 3.28	24.40 ± 6.13**	32.26 ± 7.71	30.39 ± 3.39	30 - 85
	females	51.31 ± 10.93	56.19 ± 16.62	48.46 ± 10.47	38.17 ± 5.40	51.76 ± 12.61	46.38 ± 10.77	30 - 85
Total Bilirubin (µmol/l)	males	2.30 ± 0.36	2.24 ± 0.27	1.90 ± 0.44	1.73 ± 0.19**	2.22 ± 0.27	2.34 ± 0.19	up to 4.1
	females	2.22 ± 0.33	2.34 ± 0.34	1.82 ± 0.27*	1.76 ± 0.25*	2.16 ± 0.25	1.80 ± 0.27**	up to 4.1
Sodium (mmol/l)	males	148.8 ± 3.2	148.1 ± 4.0	149.3 ± 4.9	145.1 ± 5.4	150.8 ± 4.3	150.9 ± 2.8	135 - 150
	females	151.4 ± 4.5	149.8 5.1	147.7 ± 5.8	144.3 ± 6.0*	148.6 ± 1.4	149.7 ± 3.1	135 - 150
Potassium (mmol/l)	males	5.74 ± 0.35	5.61 ± 0.31	5.68 ± 0.44	5.11 ± 0.54*	5.68 ± 0.64	5.39 ± 0.44	4.5 - 7.0
	females	5.24 ± 0.44	5.24 ± 0.51	5.24 ± 0.68	4.68 ± 0.23	5.27 ± 0.44	4.81 ± 0.38*	4.5 - 7.0
Calcium (mmol/l)	males	2.84 ± 0.08	2.72 ± 0.13	2.57 ± 0.11***	2.61 ± 0.14**	2.82 ± 0.18	2.75 ± 0.08	2.2 - 3.0
	females	2.72 ± 0.11	2.64 ± 0.16	2.66 ± 0.08	2.76 ± 0.13	2.77 ± 0.05	2.67 ± 0.13*	2.2 - 3.0
Chloride (mmol/l)	males	103.2 ± 1.8	100.4 ± 2.4	102.8 ± 3.0	98.3 ± 2.9**	106.0 ± 3.0	106.3 ± 1.3	95 - 110
	females	107.3 ± 3.1	105.1 ± 2.7	104.4 ± 4.0	99.9 ± 3.7***	108.1 ± 1.1	108.1 ± 1.4	95 - 110
Na/K ratio	males	25.982 ± 1.222	26.459 ± 1.574	26.380 ± 1.500	28.588 ± 2.142*	26.806 ± 2.642	28.140 ± 2.059	20 - 30
	males	29.064 ± 2.492	28.772 ± 2.306	28.488 ± 2.711	30.866 ± 1.468	28.258 ± 2.225	31.282 ± 2.343	20 - 30
*p < 0.05
** p < 0.1
***p < 0.001
# p < 0.01 (U-Test)
Table 6: Liver and kidney weights (mean values and standard deviations)
Group		Control	5 mg/kg	50 mg/kg	500 mg/kg	control recovery	500 mg/kg recovery
Liver weights	males	16.669 ± 1.337	17.299 ± 2.342	16.272 ± 1.396	18.894 ± 1.367***	19.252 ± 2.712	16.379 ± 2.567
(not corrected)	females	10.771 ± 1.473	9.748 ± 1.518	10.550 ± 1.526	14.316 ± 1.939***	9.664 ± 1.367	10.418 ± 0.970**
Liver weights	males	16.150 ± 1.249	16.591 ± 1.462	16.515 ± 0.832	20.263 ± 1.129***	18.569 ± 1.197	17.061 ± 1.283
(corrected)	females	10.689 ± 1.186	9.675 ± 1.506	10.710 ± 0.560	14.309 ± 0.566***	9.552 ± 0.812	10.530 ± 0.458**
Kidney weights	males	3.156 ± 0.200	3.263 ± 0.188	3.029 ± 0.264	3.371 ± 0.332**	3.307 ± 0.338	3.272 ± 0.285
(not corrected)	females	2.127 ± 0.254	2.030± 0.245	2.184 ± 0.405	2.398 ± 0.307	2.027 ± 0.286	2.145± 0.254
Kidney weights	males	3.085 ± 0.171	3.166 ± 0.165	3.000 ± 0.250	3.558 ± 0.201**	3.255 ± 0.299	3.323 ± 0.232
(corrected)	females	2.112 ± 0.284	2.017 ± 0.190	2.213 ± 0.193	2.397 ± 0.170	2.004 ± 0.146	2.168 ± 0.209
*p < 0.05 (Scheffé test)
** p < 0.1 (Scheffé test)
***p < 0.001 (Scheffé test)

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for repeated dose toxicity was considered to be 50 mg/kg bw/d based on effects on liver weights and bilirubin level. All other findings in haematology, clinical chemistry and histopathology are considered to be coincidental and/or of no biological importance as they were completely reversible after a recovery period of 4 weeks.

Executive summary:

The potential toxicity of benzyltouene was evaluated following repeated oral administration for 13 weeks, according to OECD Guideline 408. Benzytoluene was administered once daily by gavage to Sprague-Dawley rats (10 Males and 10 Females), at the dose-levels of 0, 5 , 50 and 500 mg/kg/d during 90 days.

Body weights were recorded pre-test, weekly and at death or prior necropsy. Animals were observed daily for toxicity and pharmacological effects, and twice daily for morbidity and mortality. Food consumption was calculated weekly. A neurobehavioral examination was performed. Whole blood, serum and plasma were sampled just prior necropsy and analysed for related haematological and clinical chemistry parameters.

All animals were examined for gross pathology, and organs were weighted and submitted to histopathology.

The No Observed Adverse Effect Level (NOAEL) for repeated dose toxicity was considered to be 50 mg/kg bw/d based on effects on liver weights and bilirubin level. All other findings in haematology, clinical chemistry and histopathology are considered to be coincidental and/or of no biological importance as they were completely reversible after a recovery period of 4 weeks.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

7

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Data for repeated dose toxicity demonstrate that no classification is required. The gap between NOAEL (50 mg/kg) and LOAEL (500 mg/kg) is slight and the effects observed at 500 mg/kg are not of very high toxicological concern (especially slight hypertrophy of hepatocytes).