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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Study conducted prior to the adoption of the most recent version of this Guideline
Deviations:
yes
Remarks:
Individual bodyweights at study start were not indicated; some parameters, as age of animals at study start and humidity level during study, were not reported; sampling time was only 6 hours following end of treatment
GLP compliance:
no
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ugilec 101
- Physical state: Clear colourless liquid

Test animals

Species:
mouse
Strain:
other: COBS CD-1 (ICR) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: no data
- Weight at arrival: 18 - 21 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, overnight
- Housing: 2 or 5 animals/sex/cage
- Diet: laboratory diet ad libitum
- Water: tap water ad libitum
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): no data
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12


IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Ugilec 101 was miscible with corn oil
- Concentration of test material in vehicle: 12.5, 25.0, 50.0 mg/mL
- Amount of vehicle (if gavage or dermal): animals in all groups were dosed with the standard volume of 0.1 ml/10 g bodyweight (10 mL/kg bw)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Ugilec 101 was prepared as a solution in corn oil using a high speed mixer.
Duration of treatment / exposure:
2 administrations separated by an interval of 24h.
Frequency of treatment:
daily
Post exposure period:
6 hours
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Justification for choice of positive control(s): yes, a known mutagen (recommanded by OECD for this specific test)
- Route of administration: intraperitoneal injection
- Doses / concentrations: 4 mg/kg for 2 days

Examinations

Tissues and cell types examined:
- Tissues: Bone marrow smear from each femur
- Cells: Erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the Maximal Tolerated Dose obtained from preliminary toxicity testing

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling was performed on dead animals sacrificed 6 hours after the last administration.

DETAILS OF SLIDE PREPARATION:
Following animal sacrifice, a direct bone marrow smear was made onto a slide containing a drop of calf serum. The slide was cleaned by immersion in methanol for 24 hours and air-dried immediately before use. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol overnight. After fixation, the smears were air-dried and placed in buffered distilled water (pH 6.8) for 10 minutes prior to staining in Giemsa (dilution factor; 1 part Giemsa : 9 parts buffered distilled water, pH 6.8) for 10 minutes. After rinsing in buffered distilled water (pH 6.8), the slides were air-dried and mounted in DPX.

METHOD OF ANALYSIS:
The stained smears were coded and examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal and the ratio of normochromatic to polychromatic erythrocytes.
Evaluation criteria:
no data
Statistics:
no data

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 500 mg/kg bw/d
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:
Phase I (2 animals/sex/dose): 2 administrations from 250 to 9700 mg/kg bw/d (from 500 to 19400 mg/kg bw in total)
Phase II (5 animals/sex/dose): 2 administrations from 500 to 1000 mg/kg bw/d (from 2000 to 4000 mg/kg bw in total)
- Mortality (see Table 1 in the field Remarks on results)
- Clinical signs of toxicity:
After administration of Ugilec 101 at 250 mg/kg bw/d, no toxic reactions were observed.
At 1000 mg/kg bw/d, a toxic reaction consisting of hypopnoea and lethargy was observed 1 hour after the first dose and the symptoms were still present 7 hours later. However on the morning of the second day (24 hours after the first dose) the symptoms were not observed, but reappeared 1 hour after the second dose and were still present at the time of sacrifice, 5 hours later.
Between 1500 and 9700 mg/kg bw/d, a toxic reaction consisting of hypopnoea, ptosis and pale extremities were observed 1 hour after the first dose. In addition, ataxia and tremors were observed 2 hours later and all the symptoms were still present 4 hours later (8 hours after the first dose). However on the morning of the second day (24 hours after the first dose) the symptoms were not observed, but reappeared 1 and 3 hours later and were still present at the time of sacrifice, 6 hours after the second dose.



RESULTS OF DEFINITIVE STUDY (see Table 2 in the field Remarks on results)
- Mortality:
No mortality was observed in the low dose animal group.
One male treated at 250 mg/kg bw/d (500 mg/kg bw in total) was found dead 24 hours after the first treatment.
One male treated at 500 mg/kg bw/d (1000 mg/kg bw in total) was found dead 2 hours after the second treatment.

- Clinical signs of toxicity:
After administration of Ugilec 101 at dosages of 125 and 250 mg/kg bw/d (250 and 500 mg/kg bw in total, respectively) no toxic reactions were observed.

Dosage of 500 mg/kg bw/d (1000 mg/kg bw in total) produced a toxic reaction consisting of hypopnoea and lethargy which was observed 1 hour after the first dose and the symptoms were still present 7 hours later. However on the morning of the second day (24 hours after the first dose) the symptoms were not observed, but reappeared 1 hour after the second dose and were still present at the time of sacrifice, 5 hours later.

Macrocopic examination at post mortem was not possible for the animal dead in the mid-dose treated group, due to visceral autolysis, and failed to reveal any abnormalities in the high-dose treated dead animal.

- Induction of micronuclei (for Micronucleus assay):
After administration of Ugilec 101 at all total dosages, the group mean micronucleated cell counts were comparable with the concurrent control value.
- Ratio of NCE/PCE (for Micronucleus assay):
After administration of Ugilec 101 at all total dosages the group mean normochromatic to polychromatic cell ratios were comparable with the concurrent control value.
- Appropriateness of dose levels and route:
Toxic reactions observed in the micronucleus test as expected as they were similar to the reactions observed in prelimnary toxicity study at the same dose levels.

Any other information on results incl. tables

Table 1 Preliminary toxicity results

Test design

Treatment (2 administrations at 24-h interval)

Mortality ratio

Males

Females

Phase I:
Pilot study

Vehicle

0/2

0/2

TS at 250 mg/kg bw/d

0/2

0/2

TS at 2500 mg/kg bw/d

2/2

2/2

TS at 5000 mg/kg bw/d

2/2

2/2

TS at 7500 mg/kg bw/d

2/2

2/2

TS at 9700 mg/kg bw/d

2/2

2/2

Phase II:
Toxicity assay

Vehicle

0/5

0/5

TS at 1000 mg/kg bw/d

4/5

4/5

TS at 1500 mg/kg bw/d

5/5

3/5

TS at 2000 mg/kg bw/d

5/5

5/5

Table 2 Micronucleus assay results

Treatment  

(2 administrations at 24-h interval)

Sacrifice time
(h)

Mean number of micronucleated cells per 2000 polychromatic erythrocytes per animal

Mean ratio of normochromatic
to polychromatic erythrocytes

Vehicle

30

1.8

1.7

TS at 125

mg/kg bw/d

30

1.9

1.6

TS at 250

mg/kg bw/d

30

1.4

1.4

TS at 500

mg/kg bw/d

30

2.2

1.7

Mitomycin C at 8 mg/kg bw/d

30

105.1

9.0

Applicant's summary and conclusion

Conclusions:
Ugilec 101 (benzyltoluene) did not present any in vivo genotoxic activity in the micronucleus test conducted in mice orally gavaged twice at 125, 250 and 500 mg/kg bw.
Executive summary:

The genotoxicity of benzyl toluene (Ugilec 101) was determined in a micronucleus test conducted in vivo with COBS CD-1 (ICR) BR mice using a similar method to the OECD Guideline 474. Solutions were administered by gavage at a constant volume of 10 mL/kg bw. Following preliminary toxicity assay, 500 mg/kg bw/d treatment for 2 days was selected as the maximum tolerated dose, based on high rates of mortality and severe clinical signs observed at higher dosages. Doses of 125, 250, and 500 mg/kg bw/d were administered by oral gavage to groups of 10 mice (5 males and 5 females), in two equal dosages, separated by an interval of 24 hours. Mitomycin C, administered intraperitoneally at a total dose level of 8 mg/kg, served as positive control. Animal were sacrificed 6 hours after the second administration, and bone marrow smears examined for the presence of micronuclei in 2000 polychromatic erythrocytes per mouse and for the ratio NCE/PCE. No significant increased mortality was observed in any treated groups. Signs of toxicity were only observed at the dose of 500 mg/kg bw/d, consisting of hypopnoea, ptosis, ataxia and tremors. The NCE/PCE ratio obtained in all treated animals was similar to that of control animals. The number of micronucleated polychromatic cells in animals given benzyltoluene was similar to that obtained in control animals. Conversely, animals treated with Mitomycin C presented a highly significant increase in the number of micronucleated polychromatic cells. Benzyl toluene was concluded to be negative in the micronucleus test with mice.