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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The test conditions are comparable to the recognised EU and OECD test guidelines. However, only one sex was tested, the GLP status is not stated, there are no historical control data (negative and positive) and the ratios PCE/NCE were not available.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
only male rats used, lack of animal housing conditions
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
p-tert-butylcatechol (TBC). No other information available.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
no data available

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: no data
Details on exposure:
Male F344/N rats were injected intraperitoneally three times at 24-hour intervals with TBC dissolved in corn oil.
Vehicle control animals were injected with corn oil only.
The positive control rats received injections of 10 mg/kg cyclophosphamide.

In the first experiment, animals were exposed to 0, 125, 250 and 500 mg/kg bw/day.
In the second experiment, because of a severe toxicity at 500 mg/kg bw/day, animals were exposed to 0, 125, 250 and 300 mg/kg bw/day.
Duration of treatment / exposure:
3 days
Frequency of treatment:
daily injections
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Trials 1 and 2
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Trials 1 and 2
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Trials 1 and 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Trial 2 only
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Trial 1 only
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: intraperitoneal
- Doses / concentrations: 10mg/kg

Examinations

Details of tissue and slide preparation:
The rats were killed 24 hours after their last injection, and blood smears were prepared from bone marrow cells obtained from the femurs. Air-dried smears were fixed and stained; 2000 polychromatic erythrocytes (PCEs) were scored for frequency of micronucleated cells in up to 5 animals per dose group.
Evaluation criteria:
An individual trial was considered positive if the trend test P value was less than or equal to 0.025 or if the P value for any single dose group was less than or equal to 0.025 divided by the number of dose groups. A final call of positive for micronucleus induction was preferably based on reproducibility positive trials (as noted above). Ultimately, the final call was determined by the test facility scientific staff after considering the results of statistical analyses, reproducibility of any effects observed, and the magnitudes of those effects.
Statistics:
The frequency of micronucleated cells among PCEs was analysed by a statistical software package that tested for increasing trend over dose groups with one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the control group.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at > 250 mg/kg in trial 1
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
In the first assay, signs of systemic toxicity were observed at the highest dose of 500 mg/kg. Only one animal (out of 5) survived. Because of this severe mortality, the dose level of 500 mg/kg was not included in statistical analysis.
In the second assay conducted up to 300 mg/kg, no signs of toxicity were noted.
In the two experiments, the PCE/NCE ratios were not presented. Considering the high mortality observed at 500 mg/kg in the first assay and the absence of effect noted at 300 mg/kg in the second assay, 4 -tert-butylpyrocatechol was tested up to the limit of toxicity.
In the two experiments, positive controls induced an appropriate response. But no historical control (either positive or negative) data were presented. Consequenlty, no information is available about the range of micronucleated PCEs expected in the negative control group.

In the first assay, a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) was noted over the two analysed doses. In the negative control, 1 ± 0.16 MNPCE were found for 1000 PCE. At 125 and 250 mg/kg, 1.80 ± 0.66 and 2.90 ± 0.83 MNPCE for 1000 PCE were observed respectively.
In the second assay, there was no statistically significant increase in the frequency of MNPCE. In the negative control, 1.80 ± 0.34 MNPCE were found for 1000 PCE . At 125, 250 and 300 mg/kg, 3.00 ± 0.69, 1.33 ± 0.17 and 2.13 ± 0.24 MNPCE for 1000 PCE were observed respectively. Although increases over the negative control were noted at 125 and 300 mg/kg, these findings were not dose-related and not statisticaly significant.

No historical control data were presented in this study. However, according to the 2 experiments, the MNPCE frequency in the negative control group could be approximately estimated over the range 1.00 - 1.80 (standard error not taken into account).

The response levels in the treated groups were similar between the two experiments. In the first assay, the MNPCE frequency observed in the treated animals were over the range 1.80 - 2.90. In the second one, they were over the range 1.33 - 3.00 without dose -related trend. Over the doses ranges tested in the 2 experiments, the response fluctuated from 1.33 up to 3.00 MNPCE per 10000 PCE, without systematically a dose-related trend. In addition, the response of statistical analysis was not reproducible over a similar response level between the two assays. The variability of the MNPCE values observed in the treated groups seems to come from a normal fluctuation of the biological response, and not to be induced by a treatment related effect.

In absence of historical control data and in absence of reproducibility in the results whether the statistical significance and the dose related trend, no clear conclusion could be drawn. There is no evidence of a treatment related induction of the MNPCE. This study is considered unconclusive. It should be noted that US Department of Health and Human Services (in the scope of the National Toxicology Program) considered this study as negative.

Any other information on results incl. tables

Systemic toxicity: Induction of micronuclei in bone marrow polychromatic erythrocytes (PCE) of male rats:

Compound

Dose (mg/kg)

Micronucleated PCEs/1000 PCEs (mean)

Variation versus controls

P value

Trial 1

 

 

 

 

Corn oil

 

1.00

 

 

4-tert-butylpyrocatechol

125

1.80

x 1.8

0.1225

 

250

2.90

x 2.9

0.0050

 

500

2.00

x 2

 

Cyclophosphamide

10

17.80

x 17.8

 

Trial 2

 

 

 

 

Corn oil

 

1.80

 

 

4-tert-butylpyrocatechol

125

3.00

x 1.7

0.0414

 

250

1.33

x 0.7

0.7610

 

300

2.13

x 1.2

0.3114

Cyclophosphamide

10

11.70

x 6.5

 

Applicant's summary and conclusion

Conclusions:
4-tert-butylpyrocatechol (TBC) induced a dose-related increase in micronuclei in bone marrow cells of rats receiving 3 daily intraperitoneal injections of 125 or 250 mg/kg (the highest dose, 500 mg/kg, was lethal to some animals).
However, no significant dose-related increase in micronuclei was observed in a second trial at doses ranging from 125 to 300 mg/kg.
In absence of historical control data and due to the lack of reproducibility in the results whether the statistical significance and the dose related trend, no clear conclusion could be drawn. This study is considered unconclusive. It should be noted that US Department of Health and Human Services (in the scope of the National Toxicology Program) considered this study as negative.
Executive summary:

The substance 4 -tert-butylpyrocatechol was tested in a rat bone marrow micronucleus assay (NTP study, 2002).

The animals received 3 daily intraperitoneal injections at 24 -hour intervals, at the dose levels of 0, 125, 250 and 500 mg/kg bw in a first assay and at 0, 125, 250 and 300 mg/kg bw in a second assay. The vehicle was corn oil.

In the first assay, signs of systemic toxicity were observed at the highest dose of 500 mg/kg. Only one animal (out of 5) survived. Because of this severe mortality, the dose level of 500 mg/kg was not included in statistical analysis.

In the second assay conducted up to 300 mg/kg, no signs of toxicity were noted.

In the two experiments, positive controls induced an appropriate response. No historical control (either positive or negative) data were presented. According to the 2 experiments, the MNPCE frequency in the negative control group could be approximately estimated over the range 1.00 - 1.80.

In the first assay, a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) was noted over the two analysed doses. In the negative control, 1 ± 0.16 MNPCE were found for 1000 PCE. At 125 and 250 mg/kg, 1.80 ± 0.66 and 2.90 ± 0.83 MNPCE for 1000 PCE were observed respectively.

In the second assay, there was no statistically significant increase in the frequency of MNPCE. In the negative control, 1.80 ± 0.34 MNPCE were found for 1000 PCE . At 125, 250 and 300 mg/kg, 3.00 ± 0.69, 1.33 ± 0.17 and 2.13 ± 0.24 MNPCE for 1000 PCE were observed respectively.

The response levels in the treated groups were similar between the two experiments. In the first assay, the MNPCE frequency observed in the treated animals were over the range 1.80 - 2.90. In the second one, they were over the range 1.33 - 3.00. Over the doses ranges tested in the 2 experiments, the response fluctuated from 1.33 up to 3.00 MNPCE per 10000 PCE, without systematically a dose-related trend. In addition, the response of statistical analysis was not reproducible over a similar response level between the two assays. The variability of the MNPCE values observed in the treated groups seems to come from a normal fluctuation of the biological response, and not to be induced by a treatment related effect.

In conclusion, there is no evidence of a treatment related induction of the MNPCE. This study is considered unconclusive.

It should be noted that US Department of Health and Human Services (in the scope of the National Toxicology Program) considered this study as negative.