Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Four Ames tests are available and three of them are considered to be reliable (reliability 1 and 2 according to Klimisch scale). These three tests showed negative results with and without metabolic activation. Therefore based on these three studies, 4-tert-butylpyrocatechol is not considered to be mutagenic in bacteria.

A chromosome aberration test conducted similar to the OECD 473 test guideline is available (CIT, 1992 – reliability 2). Under the experimental conditions, 4-tert-butylpyrocatechol did not showed any increase of chromosomal aberrations in Chinese Hamster Ovary (CHO) cells treated up to the limit of an acceptable cytotoxicity and under an exposure time of 3 hours, either in the absence or in the presence of metabolic activation.

Regarding mutations in mammalian cells, one publication (McGregor et al., 1988 – reliability 2) similar to the OECD 476 test guideline gives ambiguous results on L5178Y mouse lymphoma cells and only the condition without metabolic activation has been tested. Under the experimental conditions of this Mouse Lymphoma Assay, 4-tert-butylpyrocatechol induced an increase in mutation frequency at concentrations of 4.5 or 5 µg/mL, together with a very high level of cytotoxicity (Relative Total Growth < or = to 10%), making the test results questionable at such concentrations. There was no evidence of mutagenicity at concentrations up to 4 µg/mL inclusive. A new mammalian cell gene mutation test with L5178Y mouse lymphoma cells has been conduction in 2017 (CharlesRiver, 2017 – reliability 1). 4-tert-butylpyrocatechol showed mutagenic effects in this vitro Mammalian Cell Gene Mutation Test. This positive result was seen only in the presence of metabolic activation and at toxic dose levels.

Therefore regarding in vitro tests, the registered substance is only positive in the new in vitro Mammalian Cell Gene Mutation test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
End of the study period: 30 April 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of male Sprague-Dawley rats liver induced with peritoneal Aroclor 1254 (= S9)
Test concentrations with justification for top dose:
- without S9: 31.25, 62.5, 125, 250 and 500 µg/plate for the 1st assay - 15.625, 31.25, 62.5, 125, and 250 µg/plate for the 2nd assay.
- with S9: 62.5, 125, 250, 500 and 1000 µg/plate for both assays.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene (0.5 µg/pl): for TA 98 and TA 1538 without S9; sodium azide (1 µg/pl): for TA 100 and TA 1535 without S9; 9-amino-acridine (50 µg/pl): for TA 1537 without S9; 2-anthramine (1 or 2 µg/pl): for all strains with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 to 72 hours at 37°C
NUMBER OF REPLICATIONS: 3








Evaluation criteria:
- a test substance is considered as mutagenic if, for each test, it induces a doubling in the number of revertants when compared to that in the negative and/or solvent controls, for at least one of the tested strains and at one or more of the tested concentrations. In this case, a statistically significant dose relationship is investigated, using a linear regression analysis, and considered as significant if p ≤ 0.05 (for n values, the correlation coefficient r must be ≥ 0.47).
- a test substance is considered as non-mutagenic if the above two criteria are not fully met.
Statistics:
linear regression analysis
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: TA 98 and TA 100 strains: ≥ 1000 µg/plate without S9, ≥ 2500 µg/plate with S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Toxicity:
In the preliminary assay, TBC was toxic to bacterial strains TA98 and TA100 stains at 1000 µg/plate or greater without S9 and 2500 µg/plate or greater with S9.
At lower concentrations, no toxicity was observed.

Mutagenic activity:
Without S9, TBC was toxic at 500 µg/plate and toxicity was also observed in the first test without S9 for the TA100 strain at some concentrations and varied according to the triplicate.
The solvent control results were equivalent to those usually obtained in the laboratory.
The number of revertants induced by the positive controls were higher than the controls, which demonstrated the sensitivity of the test.
The number of revertants obtained in the presence of TBC with and without S9 for the 5 strains was equivalent to that of the negative controls.

First assay(number of revertants/plate)

 

TA 1535

TA 1537

TA 1538

Conc.

µg/plate

-S9

Cytotoxic

-S9

Cytotoxic

-S9

Cytotoxic

0*

10

no

1

no

2

no

31.25

8

no

3

no

2

no

62.5

4

no

3

no

7

no

125

9

no

5

no

6

no

250

6

no

3

no

3

no

500 (1)

t

yes

1

yes

4

yes

Positive control

10

no

35

no

22

no

 

TA 98

TA 100

 

 

Conc.

µg/plate

-S9

Cytotoxic

-S9

Cytotoxic

 

0*

2

no

8

no

 

31.25

4

no

27

yes

 

62.5

7

no

6

yes

 

125

7

no

19

no

 

250

2

no

15

yes

 

500 (1)

3

yes

25

yes

 

Positive control

8

no

15

no

 

*solvent control with DMSO

t: toxicity

(1): yellow colouration

Second assay(number of revertants/plate)  

 

TA 1535

TA 1537

TA 1538

Conc.

µg/plate

-S9

Cytotoxic

-S9

Cytotoxic

-S9

Cytotoxic

0*

14

no

10

no

23

no

15.625

16

no

13

no

18

no

31.25

17

no

13

no

15

no

62.5

17

no

9

no

24

no

125

10

no

13

no

25

no

250

11

no

8

no

25

no

Positive control

232

no

120

no

292

no

 

TA 98

TA 100

 

Conc.

µg/plate

-S9

Cytotoxic

-S9

Cytotoxic

 

0*

24

no

118

no

 

15.625

29

no

112

no

 

31.25

27

no

117

no

 

62.5

21

no

93

no

 

125

27

no

120

no

 

250

19

no

61

no

 

Positive control

255

no

384

no

 

*solvent control with DMSO

First assay(number of revertants/plate)

 

TA 1535

TA 1537

TA 1538

Conc.

µg/plate

+S9

Cytotoxic

+S9

Cytotoxic

+S9

Cytotoxic

0*

15

no

1

no

5

no

62.5

11

no

3

no

0

no

125

10

no

2

no

4

no

250

9

no

3

no

1

no

500

7

no

1

no

2

no

1000

6

no

1

no

2

no

Positive control

231

no

12

no

48

no

 

TA 98

TA 100

 

 

Conc.

µg/plate

+S9

Cytotoxic

+S9

Cytotoxic

 

0*

5

no

26

no

 

62.5

2

no

22

no

 

125

4

no

7

no

 

250

7

no

10

no

 

500

4

no

16

no

 

1000

3

no

5

no

 

Positive control

27

no

106

no

 

*solvent control with DMSO

Second assay(number of revertants/plate)

 

TA 1535

TA 1537

TA 1538

Conc.

µg/plate

+S9

Cytotoxic

+S9

Cytotoxic

+S9

Cytotoxic

0*

17

no

13

no

25

no

62.5

21

no

11

no

23

no

125

22

no

15

no

24

no

250

18

no

14

no

31

no

500

16

no

13

no

22

no

1000

13

no

12

no

29

no

Positive control

178

no

225

no

688

no

 

TA 98

TA 100

 

Conc.

µg/plate

+S9

Cytotoxic

+S9

Cytotoxic

 

0*

34

no

112

no

 

62.5

32

no

126

no

 

125

29

no

112

no

 

250

34

no

95

no

 

500

23

no

100

no

 

1000

25

no

90

no

 

Positive control

1250

no

1283

no

 

*solvent control with DMSO

Conclusions:

Negative without metabolic activation
Negative with metabolic activation

Under the experimental conditions, 4-tert-butylpyrocatechol did not show mutagenic activity in the Ames test up to 1000 µg/plate with metabolic activation and up to 5250 µg/plate without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria (CIT study 7682 MMO, 1992), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to 4 -tert-butylpyrocatechol (TBC) (purity: 99%), in DMSO at concentrations of 31.25, 62.5, 125, 250 and 500 µg/plate (first assay) or at concentrations of 15.625, 31.25, 62.5, 125 and 250 µg/plate (second assay) in the absence of mammalian metabolic activation (rat liver S9 fraction) and at concentrations of 62.5, 125, 250, 500 and 1000 µg/plate (both assays) in the presence of mammalian metabolic activation (rat liver S9 fraction) (plate incorporation method).

4 -tert-butylpyrocatechol was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. Therefore, TBC was considered not mutagenic in the Ames test up to 1000 µg/plate with metabolic activation and up to 250 µg/plate without metabolic activation (the 500 µg/plate concentration was cytotoxic).

This study is classified as acceptable. This study satisfies the requirements of the test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
End of the study period: 25 August 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No long term treatment (exposure for 24h) was conducted in the absence of metabolic activation as recommended by the guideline OECD 473. Only short term treatments (3 h) with and without metabolic activation were conducted. Only 2 dose levels analysed for the genotoxicity had an acceptable cytotoxicity. The third dose analysed had a reduction of mitotic index by 80%.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
mammalian cell line, other: Chinese hamster ovary (CHO) cells
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rats liver induced with Aroclor 1254 (= S9)
Test concentrations with justification for top dose:
0, 2.5, 5, 7.5, 10 and 20 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (without S9); benzo(a)pyrene (with S9)
Details on test system and experimental conditions:
The assay using duplicate cultures was performed both with and without S9. A single sampling time was used: 24 hours, i.e. 1.5 cell cycle times from the beginning of treatment.

For each culture, approximately 5 x 10E5 cells were seeded in 3 mL DMEM (Eagle medium modified by Dulbecco) medium containing 10% fetal calf serum, 1% L. glutamine, penicillin, streptomycin and fungizon in flasks of 25 cm2. The flasks were then placed at 37°C in a humidified atmosphere of 5% CO2 in 95% air. After 24 hours, the cultures were exposed to the test subtance:
- for 3 hours at 37°C in culture medium without fetal calf serum for the assay without S9,
- for 3 hours at 37°C in the culture medium without fetal calf serum containing 15% S9, for the assay with S9.
After treatment, the cells were observed under a microscope to assess any morphological alterations, the treatment medium removed, the cells rinsed and fresh medium added. The cultures were then incubated for 21 hours at 37°C.
Two hours before cell collection, the cells were treated with a colcemid solution in order to stop mitosis at the metaphase-stage.
The cells were then rinsed, trypsinated, harvested and centrifuged. After a hypotonic treatment, they were fixed in a methanol/acetic acid mixture, spread on slides and stained with Giemsa. Two slides/culture were prepared.

Evaluation criteria:
- a test substance was considered as clastogenic if, at one or more concentrations, it induced a statistically significant increase in the incidence of cells with aberrations. This increase should exceed the normal range of historical negative control data.
- a test substance was considered as non-clastogenic if there was no significant increase in cells with aberrations frequency at any doses, above concurrent control frquencies.

Evaluation of the results:
The mitotic index was evaluated for all the cultures. The chromosomal abnormalities were scored on the slides corresponding to 3 treated cultures which were selected according to the mitotic index and to all the controls. 200 metaphases/concentration were analysed whenever possible. The structural aberrations (chromatid and chromosomal gaps, deletions and exchanges and others (multiple aberrations and pulverizations)) were recorded for each metaphase.
Statistics:
The incidence of cells wtih aberrations (excluding gaps) in treated cultures was compared to that of the solvent cultures using the "Chi-square" test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 10 µg/mL and 20 µg/mL, a reduction of the mitotic index of 80% and 100% was observed, respectively. No significant reduction was noted at 7.5 µg/mL and below.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 10 and 20 µg/mL, a reduction of the mitotic index of 40% and 80% was observed, respectively. At 7.5 µg/mL and below, no significant reduction was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Cytotoxicity:
The mitotic index was, as compared to the controls:
- without S9: reduced by 100% at 20 µg/mL, by 80% at 10 µg/mL, by 13% at 7.5 µg/mL, by 40% at 5 µg/mL and by 27% at 2.5 µg/mL;
- with S9: reduced by 80% at 20 µg/mL, by 40% at 10 µg/mL and similar to that of controls at the lower concentrations (by 0% at 7.5 µg/mL, by 20% at 5 µg/mL and by 27% at 2.5 µg/mL)

Genotoxicity:
The chromosomal abnormalities were scored on the slides corresponding to the 3 following concentrations:
- without S9: 2.5, 5 and 10 µg/mL
- with S9: 5, 10 and 20 µg/mL.

It should be noted that the dose levels of 10 µg/mL in the absence of metabolic activation, and 20 µg/mL in the presence of metabolic activation, showed a very severe cytotoxicity (reduction of the mitotic index of 80%). Indeed, these 2 tested dose levels should not have been selected for a reliable genotoxicity analysis. According to the recent guideline OECD 473, the highest tested concentration should achieve 55 ± 5% cytotoxicity using the recommended cytotoxicity parameters (i.e. reduction in Mitotic Index), and care should be taken in interpreting positive results only to be found in the higher end of this 55 ± 5% cytotoxicity range.
In addition, no analysis was conducted on the dose level of 7.5 µg/mL (probably because the mitotic index is the less reducted at this dose).
The absence of the analysis at the dose of 7.5 µg/mL did not affect the reliability of the study. The analysed dose range was narrow with a concentration interval of 2 fold between the analysed doses (2.5, 5 and 10 µg/mL without S9 mix and 5, 10 and 20 µg/mL with S9 mix). In addition, no cytotoxicity was observed at 7.5 µg/mL and below, and no genotoxicity was noted over the analysed range.

The analysis of the chromosomal aberrations gave the following results:
- With S9, the incidence of cells with aberrations in the cultures treated with TBC was similar to that of the negative controls, whatever the analysed dose.
- Without S9, at 2.5 and 5 µg/mL, the incidence of cells with aberrations was similar to that of the controls.
At 10 µg/mL, a statistically significant increase (p<0.05) was observed when compared to the solvent control and out of the the historical data. but this increase was not statistically significant when compared to the untreated cultures. In addition, as discussed above, this positive effect was observed at the dose level of 10 µg/mL showing a mitotic index reduced by 80%, i.e. very above the maximum cytotoxicity recommended by the guideline. This severe cytotoxicity of 80% does not allow a reliable genotoxicity analysis, and the genotoxicity analysis should not be considered.
Over the analysed range from 2.5 to 5 µg/mL (showing an acceptable cytotoxicity), no increase in the cells with aberrations was observed.

The incidence of cells with aberrations in the negative and solvent controls was within the range of the historical data.

The incidence of cells with aberrations in the positive controls was significantly higher (p<0.001) than that in the control cultures, indicating the sensitivity of the test system.

In conclusion, according to the recent guideline OECD 473 (adopted on 29 july 2016), no increase in the cells with aberrations was observed in the tested dose levels with an acceptable cytotoxicity, either in absence or in presence of metabolic activation.
Remarks on result:
other: A weak positive effect was observed at 10 µg/mL. The cytotoxicity was very severe at this dose (reduction of the mitotic index by 80%). According to the recent guideline, this dose level should not have been selected for a reliable genotoxicity analysis.
Conclusions:
Under the experimental conditions, 4-tert-butylpyrocatechol did not showed any increase in the chromosomal aberrations test performed in Chinese Hamster Ovary (CHO) cells treated up to the limit of an acceptable cytotoxicity and under an exposure time of 3 hours, either in the absence or in the presence of metabolic activation.
Executive summary:

In a mammalian cell cytogenetics assay, CHO (Chinese Hamster Ovary) cell cultures were exposed for an exposure time of 3 hours to 4 -tert-butylpyrocatechol (TBC) (purity: 99%) in DMSO at concentrations of 0, 2.5, 5, 7.5, 10 and 20 µg/mL with and without metabolic activation (rat liver S9 fraction).

In the absence of metabolic activation, the test substance showed severe cytotoxic effects as evaluated by mitotic index which was reduced by 80% compared to the control at 10 µg/mL and by 100% at 20 µg/mL. The dose selected for the genotoxicity effect were 10, 5 and 2.5 µg/mL. At 10 µg/mL, a statistically significant increase was observed when compared to the solvent control and it was out of the the historical data.

But this increase was not statistically significant when compared to the untreated cultures. In addition, this positive effect was only observed at the dose level of 10 µg/mL showing a mitotic index reduced by 80%, i.e. very above the maximum cytotoxicity recommended by the guideline. This severe cytotoxicity of 80% does not allow to retain this tested dose for the genotoxicity analysis.

In absence of S9, the genotoxicity analysis over the range showing an acceptable cytotoxicity (2.5 to 5 µg/mL) did not show any increase in the cells with aberrations.

In the presence of metabolic activation, the test substance showed cytotoxic effects at 20 µg/mL as seen by a decrease in mitotic index of 80% compared to the control. The analysis of the clastogenic effect was done at 5, 10 and 20 µg/mL. No increase in the cells with aberrations was noted.

Positive controls induced the appropriate response.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data (In vitro chromosomal analysis in CHO cells).

Under the experimental conditions, 4-tert-butylpyrocatechol did not showed any increase in the chromosomal aberrations test performed in Chinese Hamster Ovary (CHO) cells treated up to the limit of an acceptable cytotoxicity according to the recent guideline OECD 473, either in the absence or in the presence of metabolic activation after 3 hours of incubation.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Even if the GLP status is not stated, the test conditions are comparable to the EC and OECD guidelines recommandations. However, no metabolic activation and no positive control were used.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
other: Mammalian cell gene mutation test
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Metabolic activation system:
none
Test concentrations with justification for top dose:
0.08, 0.24, 0.8, 2.4 and 8 µg/mL in the first assay
1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 µg/mL in the second assay
Details on test system and experimental conditions:
CELLS:
tk+tk- 3.7.2.C heterozygote of L5178Y mouse lymphoma cells, obtained from Dr. D. Clive, Burroughs Wellcome Company, Research Triangle Park, NC.
Samples were cultured and used for up to 3 months, then discarded.
Laboratory cultures were confirmed as free of mycoplasma and maintained in Fisher's medium at 37°C. Fisher's medium was supplemented with 2 mM L-glutamine, sodium pyruvate, 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (designated as F10P).

EXPOSURE:
Each exposed culture consisted of 6 x 10E6 cells in a final volume of 10 mL F5P (F10P, but contaning only 5% serum). Superoxide dismutase (SOD) or catalase was added, to give a concentration of 100 units/mL.
The tubes were incubated for 4 h, sedimented by centrifugation (500g for 10 min), washed, and finally resuspended in 20 mL F10P.
These cell suspensions (3 x 10E5 cells/mL) were incubated for 2 days. After 48 h, cell density was adjusted to 2 x 10E5 cells/mL.

CLONING EFFICIENCY:
A 0.1 mL sample of the cell suspension was withdrawn and diluted to 1:100.
Three 0.1-mL samples of the diluted cultures were mixed with 25 mL cloning medium (Fischer' medium containing 20% heat-inactivated horse serum = F20P) containing 0.35% Noble agar, and poured into Petri dishes. Each plate contained 200 cells.

MUTANT SELECTION:
3 aliquots (10E6 cells) of the remaining culture were mixed with 20 mL F20P to give a final concentration of 0.35% Noble agar and 3 µg trifluorothymidine/mL, then poured into Petri dishes.

INCUBATION:
The agar was gelled at 4°C for 5-10 min, then the plates were incubated for 11-14 days on 5% CO2:95% air at 37°C.

Colonies were counted with an automated colony counter.

CALCULATIONS:
* Relative Total Growth (RTG) = (total suspension growth x cloning efficiency) in dosed culture / (total suspension growth x cloning efficiency) in control culture

* Mutation Fraction (MF) = 200 x (mutant number per plate / viable number per plate)


Evaluation criteria:
A significant response was defined as a 2-fold increase in MF at 1 of the 3 highest dose levels, where the RTG was > 1% and mutant number was increased over the vehicle control value.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified

Mutagenicity of TBC:

 

- First assay:

Concentration (µg/mL)

CE

RTG

MF

F0P

81

99

38

F0P

61

101

39

0.08

27

37

121

0.24

23

22

161

0.8

19

24

147

2.4

15

20

147

8.0

TOX

-

-

 F0P: Culture medium (negative control)      CE: cloning efficiency

RTG: relative total growth             MF: mutation frequency

- Second assay:

Concentration (µg/mL)

CE

RTG

MF

F0P

66

93

58

F0P

77

107

42

1.0

63

74

73

1.5

62

96

77

2.0

76

94

83

2.5

556

77

75

3.0

60

70

69

3.5

67

76

55

4.0

50

38

93

4.5

36

10

290

5.0

36

4

200

 F0P: Culture medium (negative control)       CE: cloning efficiency

RTG: relative total growth             MF: mutation frequency

Conclusions:

Ambiguous without metabolic activation : increased mutation frequency together with very high toxicity at >= 4.5 µg/mL

Under the experimental conditions of this Mouse Lymphoma Assay, 4-tert-butylpyrocatechol induced an increase in mutation frequency at concentrations of 4.5 or 5 µg/mL, together with a very high level of cytotoxicity (Relative Total Growth < or = to 10%), making the test results questionable at such concentrations. There was no evidence of mutagenicity at concentrations up to 4 µg/mL inclusive.
Executive summary:

In a mammalian cell gene mutation assay, L5178Y mouse lymphoma cells cultured in vitro were exposed to 4 -tert-butylpyrocatechol at concentrations of 0.08, 0.24, 0.8, 2.4 or 8 µg/mL in a first assay and 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 µg/mL in a second assay, in the absence of mammalian metabolic activation.

In the first assay, the mutation frequency was increased over controls at all analyzable concentrations, with no clear concentration-response relationship. In the second assay, an increase of the mutation frequency was observed at 4.5 and 5 µg/mL, with no relationship to the concentration, together with a very high level of cytotoxicity (Relative Total Growth of 10% and 4%, respectively).

The authors considered this response as positive at the time of the publication. But, taking account of the current standards and recommendations on the Mouse Lymphoma Assay, such levels of cytotoxicity make those test concentrations inappropriate for assessment. No evidence of gene mutation in the absence of metabolic activation was seen up to 4 µg/mL inclusive.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells.
Target gene:
thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED

- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in OECD guidelines.
- Methods for maintenance in cell culture: Cell density was kept below 1 x 10E6 cells/ml.
- The cultures were checked for Mycoplasma contamination.
- Stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Cleansing: the mouse lymphoma cells were cleansed to reduce the amount of spontaneous mutants. They were grown for 1 day in medium containing hypoxanthine, aminopterine and thymidine, followed by a recovery period of 2 days on medium containing hypoxanthine and thymidine only. After this period cells were returned to growth medium for at least 1 day before starting the experiment.

MEDIA USED

> Growth medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin and sodium pyruvate and L-glutamin, supplemented with 10% (v/v) heat-inactivated horse serum.
> Exposure medium for 3 hour exposure: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin and sodium pyruvate and L-glutamin, supplemented with 5% (v/v) heat-inactivated horse serum.
> Exposure medium for 24 hour exposure: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin and sodium pyruvate and L-glutamin, supplemented with 10% (v/v) heat-inactivated horse serum.
> Selective medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin and sodium pyruvate and L-glutamin, supplemented with 20% (v/v) heat-inactivated horse serum and trifluorothymidine (TFT).
> Non-selective medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin and sodium pyruvate and L-glutamin, supplemented with 20% (v/v) heat-inactivated horse serum.

ENVIRONMENTAL CONDITIONS

- All incubations were carried out in a humid atmosphere (80 - 100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C.
- Temperature and humidity were continuously monitored throughout the experiment.
- The CO2 percentage was monitored once on each working day.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate), prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
In order to select appropriate dose levels for mutagenicity testing, a dose-range finding test, L5178Y mouse lymphoma cells was conducted with a test item concentration up to 1662 µg/mL (or 0.01 M) in the absence and presence of S9-mix with a 3 hour treatment period.
In the absence of S9-mix, severe toxicity was observed at 32 µg/mL and above.
In the presence of S9-mix, severe toxicity was observed at 100 µg/mL and above.
Vehicle / solvent:
The test item was dissolved in dimethyl sulfoxide.
Negative solvent / vehicle controls:
yes
Remarks:
The negative control was dimethyl sulfoxide, the vehicle of the test item.
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension
- Cell density at seeding: 10E6 cells/mL

DURATION
- Exposure duration: 3 hour treatment period,in the absence as well as in the presence of S9-mix.
- Expression time (cells in growth medium): 2 days after the treatment period
- Selection time (incubation with a selection agent): 11 or 12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF CELLS EVALUATED: For determination of the mutation frequency (MF) a total number of 9.6 x 10E5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10E5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity was evaluated by the calculation of the Relative Total Growth (RTG)
- Since the maximum concentration is based on cytotoxicity, the highest concentration should aim to achieve between 20 and 10% RTG. The cultures below 10% RTG will not analysed for the mutagenicity assessment.
Evaluation criteria:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) The suspension growth over the 2-day expression period for the solvent controls should be between 8 and 32.
d) The positive control should demonstrate an absolute increase in the total mutation frequency (MF), that is, an increase above the spontaneous background MF (induced MF = IMF) of at least 300 x 10E-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10E-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10E-6).

A test item will be considered mutagenic if:
> it induces a MF of more than MF of controls + 126 (= global evaluation factor or GEF = positive threshold) and in a dose-dependent manner.
> in addition, any increase of the MF should be evaluated for its biological relevance including comparison of the results with the historical control data range.

A Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction (ToxRat Professional v 3.2.1).

A test item is considered equivocal (questionable) if no clear conclusion for positive or negative result can be made after an additional confirmation study.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH at a concentration of 1662 μg/ml was 7.2 (compared to 7.2 in the solvent control).
- Effects of osmolality: The osmolarity at a concentration of 1662 μg/ml was 0.425 Osm/kg (compared to 0.447 Osm/kg in the solvent control).
- Precipitation: 4-tert-butylpyrocatechol precipitated in the exposure medium at the top dose of 1662 μg/ml at the start of the treatment. After 3 hours of treatment, no precipitation of the test item in the exposure medium was seen anymore. The concentration used as the highest test item concentration for the dose-range finding test was 1662 μg/ml.

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test, cells were treated with a test item concentration range of 125 to 1662 µg/ml in the absence and presence of S9-mix with a 3 hour treatment period. Severe toxicity was observed at 125 µg/ml (the lowest tested dose level) in the absence and in the presence of S9-mix after the 24 hour of subculture. Therefore this experiment was ceased and a repeat experiment was performed.
In the second assay, cells were treated with a test item concentration range of 0.32 to 100 µg/ml in the absence and in the presence of S9-mix with a 3 hour treatment period.
In the absence of S9-mix, the relative suspension growth was 19% at the test item concentration of 10 μg/ml compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at test item concentrations of 32 μg/ml and above.
In the presence of S9-mix, the relative suspension growth was 22% at the test item concentration of 32 μg/ml compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at the test item concentration of 100 μg/ml.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (95%))
- Positive historical control data:
> 3 hour treatment, without S9 mix: mutation frequency range = 289 - 1425 10E6 survivors (mean = 857 +- 246 10E6 survivors)
> 24 hour treatment, without S9 mix: mutation frequency range = 253 - 1124 10E6 survivors (mean = 688 +- 187 10E6 survivors)
> 24 hour treatment, without S9 mix: mutation frequency range = -493 - 4214 10E6 survivors (mean = 1710 +- 815 10E6 survivors)

- Negative (solvent/vehicle) historical control data:
> 3 hour treatment, without S9 mix: mutation frequency range = 37 - 135 10E6 survivors (mean = 86 +- 23 10E6 survivors)
> 24 hour treatment, without S9 mix: mutation frequency range = 28 - 135 10E6 survivors (mean = 81 +- 26 10E6 survivors)
> 3 hour treatment, with S9 mix: mutation frequency range = 28 - 145 106 survivors (mean = 87 +- 28 10E6 survivors)

FIRST MUTAGENICITY TEST

Based on the results of the dose-range finding test, the dose-range tested without metabolic activation was 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, 20, 30, 40 and 50 µg/mL exposure medium. The dose levels selected to measure mutation frequencies were 0.01 to 10 μg/ml. The dose levels above 10 µg/ml were not used because of severe toxicity.

With S9-mix, the dose range tested was 1.25, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/ml exposure medium. The dose levels selected to measure mutation frequencies were 1.25 to 40 µg/mL. The dose levels above 40 µg/ml were not used because of severe toxicity.

First experiment, without metabolic activation, 3-hours treatment:

 Dose

(µg/mL)

 RTG

(%)

 Mutation frequency per

106survivors

(total colonies)

  Mutation frequency per

106survivors

(small colonies)

  Mutation frequency per

106survivors

(large colonies)

 Fold increase of the MF for total colonies (over the mean MF of the solvent controls)

 Solvent control 1 100 102  66 30  -
 Solvent control 2 100 136  70 59 

-

 0.01 106 98  53  41   0.8
 0.05 100

94 

46  44   0.7
 0.1 108 95 32  59   0.7
 0.5 118 95  36  53   0.7
 1 115 80  26  51   0.6
 2.5 101 99  44  50   0.8
 5 39 115  46  63   0.9
 10 9 153  63  80   1.2

 Positive control

(MMS)

47  1089  295  641   9.1

Note: all calculations were made without rounding off.

In the absence of S9-mix, the two highest analysed dose levels of 5 and 10 µg/mL showed a RTG of 9% (very severe toxicity) and 39% respectively. No significant increase in the mutation frequency was observed. The numbers of small and large colonies in treated cultures were comparable to the numbers of small and large colonies of the solvent controls. An increase above the 95% control limit was observed at the top dose of 10 µg/ml (153 per 106survivors). Although this increase was above the historical control data range, no dose-related was observed. In addition, this increase was below the positive threshold of 245 per 106survivors (= mean mutation frequency in the negative control + 126 per 106survivors) and corresponded only to 1.2-fold the negative control.

Therefore this increase is considered to be not biologically relevant.

First experiment, with metabolic activation, 3-hours treatment:

 Dose

(µg/mL)

 RTG

(%)

 Mutation frequency per

106survivors

(total colonies)

  Mutation frequency per

106survivors

(small colonies)

  Mutation frequency per

106survivors

(large colonies)

 Fold increase of the MF for total colonies (over the mean MF of the solvent controls)

 Solvent control 1 100 132  81 43  -
 Solvent control 2 100 135  73 54  -

1.25

107 155  79  65  1.1
 2.5 98

121

54 59  0.9
5 87 172 86  74 1.2
10 111 113 61 45 0.8
 20 46 164 80  70  1.2
 30 29 186 94 73  1.3
 40 14 281 146 100  2.1

 Positive control

(CP)

15 2909 1373 859  21.8

Note: all calculations were made without rounding off.

In the presence of S9-mix, the dose level of 40 µg/mL induced high cytotoxicity (RTG = 14%) and the mutation frequency (281 per 106survivors) is above the positive threshold of 260 per 106survivors (= mutation frequency in the negative control + 126 per 106survivors). This induction corresponds to up to 2.1-fold increase in the mutation frequency.

The test item showed to 1.9 - and 2.1 -fold increases in the MF of the small and large colonies respectively, compared to the mean mutation frequency of the small and large colonies of the negative controls.

No increase above the positive threshold was observed at any others dose levels.

Based on this experiment, no clear conclusion can be drawn since a positive result was only observed at a high cytotoxic dose and since no dose-effect relationship was observed. Therefore an additional experiment has been conducted.  

SECOND MUTAGENICITY TEST

A second mutation experiment was performed in the presence of S9 -mix with a 3 hour treatment period with duplicate cultures. The tested dose levels were 1.25, 2, 5, 10, 20, 25, 30, 35, 40, 45, 50 and 60 µg/mL exposure medium.

The dose levels selected to measure mutation frequencies were 1.25 to 40 µg/mL exposure medium (except the dose level of 2 µg/mL not judged needed and relevant for mutation frequency measurement). The dose levels above 40 μg/ml were not used for mutation frequency measurement because of severe toxicity. The dose levels of 1.25 to 10 μg/mL showed no cytotoxicity.

Second experiment, with metabolic activation, 3-hours treatment:

 Dose

(µg/mL)

 RTG

(%)

 Mutation frequency per

106survivors

(total colonies)

  Mutation frequency per

106survivors

(small colonies)

  Mutation frequency per

106survivors

(large colonies)

 Fold increase of the MF for total colonies (over the mean MF of the solvent controls)

 Solvent control 1 100 94 32 58 -
 Solvent control 2 100 94 28 63 -
 1.25 143 66  8 57   0.7
 1.25 122 74  21 51   0.7
 5 99 83  43 37   0.8
 5 95 112  48 58   1.1
 10 84 107  54 49   1.1
 10 85 107  55 47   1.1
 20 39 223  109 95   2.3
 20 41 196  84 94   2.0
 25 27 216  45 156   2.3
 25 27 225   56 156   2.3
 30 23 214  80 114   2.2
 30 15 198  67 113   2.1
 35 17 265  105 134   2.8
 35 17 313  139 138   3.3
 40 11 379  170 158   4.0
 40 11 386  193 146   4.1

Positive control

(CP) 

11 2241  1147 682   23.8

Note: all calculations were made without rounding off.

The highest tested dose level (40 µg/mL) showed a RTG of 11% when compared to the Solvent control.

The test item induced an up to 4.1 -fold increase in the mutation frequency. For the two highest doses analysed of 35 µg/mL (MF = 289 per 106survivors) and of 40 µg/ml (MF = 383 per 106survivors), the mean MF is higher than the positive threshold of 220 per 106survivors (= mutation frequency in the negative control + 126 per 106survivors).

At 30 µg/ml the MF is not higher than the postive threshold, but higher than the upper limit of the historical solvent control data range (145 x 10-6). At 20 and 25 µg/mL, the MF is higher than the postive threshold, however increases were only seen at one of the duplicate cultures and are very close to the threshold.

The test item showed up to 6.1- and 2.5-fold increases in the mutation frequency of the small and large colonies, respectively, compared to the mean mutation frequency of the small and large colonies of the solvent controls.

Based on this experiment, since a significant increase was observed at the two highest analysed dose levels, the test item was considered to induce a significant increase of the mutation frequency. 

Conclusions:
4-tert-butylpyrocatechol showed mutagenic effects in the vitro Mammalian Cell Gene Mutation Test conducted according to the OECD 490 with L5178Y Mouse Lymphoma Cells.
This positive result was seen only in the presence of metabolic activation and at toxic dose levels.
Executive summary:

The mutagenic potential of 4-tert-butylpyrocatechol was evaluated by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells. This study was conducted according to the OECD 490 guideline.

The test was performed with a 3 hour treatment period, first in the absence and in the presence of metabolic activation (S9 -mix) with a single culture (first experiment) and then, only in the presence of S9-mix with duplicate cultures (second experiment).

In the two experiments, the mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test item was dissolved in dimethylsulfoxide. No precipitate was observed in the culture medium up to and including the highest analysed dose level of 40 µg/ml.

In the first experiment, the test item was tested up to concentrations of 10 µg/mL in the absence of S9-mix and 40 µg/ml in the presence of S9-mix. Relative total growth (RTG) was 9 and 14% in the absence and presence of S9-mix, respectively.

In the absence of S9-mix, none of the tested concentrations reached the positive threshold of 245 per 106 survivors (mean mutation frequency of the controls + 126).

In the presence of S9-mix, the mutation frequency observed at 40 µg/ml, with a RTG of 14%, was 281 per 106survivors, above the positive threshold of 260 per 106survivors and outside of the historical control data range. This response corresponded to an increase of 2.1-fold the negative control. The mutation frequency observed in the second highest dose level of 30 µg/ml (with a RTG of 29%) was 186 per 106survivors, below the positive threshold.

To verify the mutagenic response observed in the first mutation experiment, an additional experiment was performed in the presence of S9-mix with duplicate cultures and an incubation time of 3 hours. In this experiment, the test item was tested up to concentrations of 40 µg/ml in the presence of S9-mix. 

At 40 µg/mL, a mean mutation frequency of 383 106per survivors was observed with a RTG of 11%, above the positive threshold of 220 106survivors and also outside the historical solvent control data range. This increase corresponded to 4.1-fold the negative control.

At 35 µg/mL (the second highest dose level), the mean mutation frequency observed was 289 106per survivors (with a RTG of 17%), also above the positive threshold and outside the historical solvent control range. The increases observed at that dose level corresponded to 2.8 -fold to 3.3-fold the negative control.

At 30 µg/mL, the mutation frequencies (214 and 198 106 survivors) were below the positive threshold of 220 106 per survivors. They were seen at a RTG between 15 and 23%. Nevertheless, they were above the historical solvent control data range.

At 25 and 20 µg/ml meaningful increases of the mutation frequency were seen. However they were above the positive threshold only at one of the duplicate cultures. They corresponded to a mutation frequency of 225 106per survivors at 25 µg/mL (with a RTG of 27%) and 223 106at 20 µg/mL (with a RTG of 39%), both just above the positive thresholf of 220 106per survivors.

In addition, a statistical significant dose related trend (p<0.001) was observed in this second experiment.

In all the lowest tested doses, no increase in the mutation frequency was observed.

Based on the results of this study and according to the positive criteria defined in the study plan, the test item is considered to be mutagenic in vitro in mammalian cells with metabolic activation. 


 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two in vivo micronucleus tests are available, one performed on mice and the other performed in rats (NTP, 2002 – reliability 2). 4-tert-butylpyrocatechol administered in feed at concentrations up to 12500 ppm for 14 weeks did not increase the frequency of micronucleated normochromatic erythrocytes in the peripheral blood of male and female mice. In addition, no significant alteration in the percentage of polychromatic erythrocytes was seen in dosed mice, indicating a lack of bone marrow toxicity. In the study in rats, 4-tert-butylpyrocatechol 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 inconclusive. However, it can be noted that the results observed in the first trial can be considered in the range of historical data for such studies. Furthermore, 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.

Based on the weight of evidence approach even if the micronucleus test in rats is doubtful (but results in range of historical data for such studies), it was not expected that the registered substance induces chromosome aberrations.

Link to relevant study records

Referenceopen allclose all

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

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

 

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.

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:
other: The test conditions and the results are sufficiently described, and are comparable to the standardised EU and OECD test guidelines. The GLP-compliance is not mentioned.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6 weeks old
- Weight at study initiation: 21.5 g (males) and 17.4 g (females)
- Fasting period before study: no
- Housing: 1 (males) or 5 (females) animals per cage (polycarbonate, changed once (male mice) or twice per week)
- Diet: NTP-2000 mash feed, available ad libidum;
- Water: tap water via automatic watering system, available ad libidum.
- Acclimation period: 15 (males) or 14 (females) days


ENVIRONMENTAL CONDITIONS
- Temperature: ca. 22°C
- Humidity: 50% +/- 15%
- Air changes: 10/hour
- Photoperiod: 12 hours light/day

IN LIFE DATES:
5 (females) or 6 (males) September 1996 to 5 (females) or 6 (males) December 1996.
Route of administration:
oral: feed
Details on exposure:
DETAILS ON DIET PREPARATION
- Rate of preparation of diet: every 4 weeks
- Type of food: irradiated feed
- Storage of food : in plastic bags inside buckets at 5°C for up to 42 days
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
7 days per week
Remarks:
Doses / Concentrations:
0, 781, 1562, 3125, 6250 and 12500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 150, 300, 635, 1300 and 2815 mg/kg bw
Basis:
other: males
Remarks:
Doses / Concentrations:
0, 135, 300, 610, 1400 and 2440 mg/kg bw
Basis:
other: females
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details of tissue and slide preparation:
At the end of the 14-week toxicity study carried out by NTP (Dunnick JK, 2002), peripheral blood samples were obtained from male and female mice.
Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with a chromatic-specific fluorescent dye mixture of Hoechst 33258/pyronin Y and coded.
Slides were scanned to determine the frequency of micronuclei in 10000 normochromatic erythrocytes (NCEs) in each of 10 animals per exposure group.
The results for NCEs in mouse peripheral and the percentage of polychromatic erythrocytes (PCEs) among the total erythrocyte population in the peripheral blood was scored for each dose group.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:

4-tert-butylpyrocatechol administered in feed at concentrations up to 12500 ppm for 14 weeks did not increase the frequency of micronucleated normochromatic erythrocytes in the peripheral blood of male and female mice. In addition, no significant alteration in the percentage of polychromatic erythrocytes was seen in dosed mice, indicating a lack of bone marrow toxicity.
Executive summary:

In a B6C3F1 mouse micronucleus assay on peripheral erythrocytes (NTP study, 2002), 10 males and females per dose were treated orally (in diet) with 4 -tert-butylpyrocatechol at doses of 0, 781, 1562, 3125, 6250 and 12500 ppm for 14 weeks. At the end of the dosing period, peripheral blood was sampled and the frequency of micronuclei was determined in erythrocytes.

There were no signs of systemic toxicity during the study. There was no significant increase in the frequency of micronucleated normochromatic erythrocytes in peripheral blood.

This study is classified as acceptable. This study was conducted comparably to the requirements of the Test Guideline OECD 474 for in vivo cytogenetic mutagenicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity:

Based on all the data from subcontracted studies and from scientific literature, 4 -tert-butylpyrocatechol was tested in an extensive battery of genetic toxicity tests, both in vitro and in vivo.

In vitro, the following genotoxic endpoints were assessed :

  • Gene mutation potential: One reverse bacterial gene mutation (Ames) test (CIT study No. 7682 MMO, 1992) was selected as a key study based on its reliability 1 according to the Klimisch cotation criteria. The results were negative both with or without metabolic activation, up to limit concentrations. These results are confirmed by 3 published studies of lower validity (cotation 2 or 3) providing negative results with or without metabolic activation. A Mouse Lymphoma Assay (McGregor, 1988) evaluating the mutagenicity in mammalian cells was also available. At the time of this experiment on several phenolic derivatives, the authors considered tert-butyl catechol as mutagenic at concentrations as low as below 10 µg/mL in their test sytem. However, according to the current standards and recommendations on the Mouse Lymphoma Assay, the highest test concentrations appear inappropriate for assessment (Relative Total Growth below or equal to 10%) and the results remain negative up to the maximum analyzable concentration (4 µg/mL) . An OECD 490 test guideline study with L5178Y Mouse Lymphoma Cells (Verspeek-Rip, 2017 - reliability 1) was conducted in order to conclude on the potency of 4 -tert butylpyrocatechol to induce mutations in mammalian cells. This test has been selected as a key study. In the first experiment, the test item was tested up to concentrations of 10 µg/mL in the absence of metabolic activation and 40 µg/ml in the presence of metabolic activation. In the absence of metabolic activation, none of the tested concentrations reached the positive threshold of 245 per 106 survivors (mean mutation frequency of the controls + 126). In the presence of metabolic activation, the mutation frequency observed at 40 µg/ml, with a RTG of 14%, was above the positive threshold and outside of the historical control data range. But at the other tested doses the positive threshold was not reached. Therefore no conclusion can be drawn in presence of metabolic activation. In order to conclude, an additional experiment was performed in the presence of metabolic activation with duplicate cultures and an incubation time of 3 hours. In this experiment, the test item was tested up to concentrations of 40 µg/ml in the presence of metabolic activation. Positive results were found at several highest doses. Based on the results of this study, the test item is considered to be mutagenic in vitro in mammalian cells with metabolic activationAnother test of low reliability (cotation 3) provided no evidence of yeast gene conversion at an unspecified concentration of 4 -tert-butylpyrocatechol.
  • Chromosomal aberration potential: One cytogenetic study in CHO cells (CIT study No. 7683 MIC, 1992) was selected as the key study based on its reliability 2 according to the Klimisch cotation criteria. The results were negative without metabolic activation, up to a limit concentration. Weak clastogenic effects were observed at the maximum concentration used with metabolic activation, together with a high level of cytotoxicity (cell survival reduced by 80%). This cytotoxicity (80%) is very above the maximum cytotoxicity recommended in the guideline. This severe cytotoxicity does not allow to retain this tested dose for the genotoxicity analysis. Therefore, without this tested dose, this test is also considered to be negative with metabolic activation. Another test of low reliability (cotation 3) provided no evidence of chromosomal aberrations in rat liver cell lines exposed for 24 hours to 4 -tert-butylpyrocatechol.

In vivo, 4 -tert-butylpyrocatechol was also evaluated for its ability to induce chromosomal aberrations in a rat bone marrow micronucleus test and a mouse erythrocyte micronucleus test (Dunnick (NTP), 2002). Both studies were key studies of validity 2 according to the Klimisch cotation criteria. The results of the mouse micronucleus test were negative following a subchronic treatment period of 14 weeks. In study in rats, 4-tert-butylpyrocatechol 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 inconclusive. It should be noted that the results in the first trial can be considered in the range of historical control for such studies and that US Department of Health and Human Services (in the scope of the National Toxicology Program) considered this study as negative.

In conclusion, it appears that 4 -tert-butylpyrocatechol is not a mutagen in bacteria. However, based on the new test performed according to the OECD 490 test guideline (Charles River, 2017), 4 -tert-butylpyrocatechol induces mutations on L5178Y mouse lymphoma cells in the presence of metabolic activation and at toxic dose levels. An in vitro chromosome aberration test was negative (CIT, 1992) and the results of the mouse micronucleus test were negative following a subchronic treatment period of 14 weeks (NTP, 2002). But, no clear conclusion can be drawn based on the micronucleus test conducted on rats (NTP, 2002). Even if in a weight of evidence approach it is not expected that the registered substance induces chromosome aberration, there is still a doubt. In order to clarify the mutagenic effect of 4 -tert-butylpyrocatechol and clear up the doubt about the inconclusive in vivo micronucleus study on rats, it is proposed to perform an additional in vivo test as a testing proposal. The comet assay appears to be the more appropriate test.

Justification for classification or non-classification

Based on the available data, no clear conclusion can be drawn on the mutagen and genotoxicity potential of 4-tert-butylpyrocatechol. In order to investigate further this point a comet assay is suggested to be performed as a testing proposal.