Phenol, 4-methyl-, reaction products with dicyclopentadiene and isobutylene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genotoxicity of phenol, 4-methyl-, reaction products with dicyclopentadiene and isobutylene was tested in cultured CHO cells (chromosomal aberration and HGPRT forward mutation assay) and in cultured S. Typhimurium cells (Ames test) in five independent studies.

Phenol, 4 -methyl-, reaction products with dicyclopentadiene and isobutylene was negative in all test systems as described above. Cellular toxicity associated with the substance was observed in cultured CHO cells, but not related to chromosomal events. The identity and quality control of the test substance was not demonstrated in any of the described studies. Assuming a 100% pure testing substance, phenol, 4 -methyl-, reaction products with dicyclopentadiene and isobutylene do not express genetic toxicity potential under the experimental conditions described.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

27 October - 5 November, 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to OECD Guideline 471 and U.S. FDA and EPA guidelines, under GLP conditions. No relevant deviations reported.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US FDA and US EPA guidelines

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene in S. Typhimurium

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Prescreen test: 50, 167, 500, 1670, 5000 µg/plate (TA 1538 and TA100 without S9)
Mutation test: 50, 167, 500, 1670, 3333, 5000 µg/plate (all strains, with and without S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: not reported

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, 2-nitrofluorene, 2-anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: one pre-screen test in duplicate, mutation assay in tripicate

DETERMINATION OF CYTOTOXICITY
- Method: counting of revertant colonies with electronic counter

Evaluation criteria:

A positive result is defined as a reproducible, statistically significant and/or dose-related increase in the number of histidine-independent colonies, with at least one dose point inducing an average revertant frequency that is two-fold that of the solvent control.

Statistics:

Significance is determined using the program developed by Moore and Felton (19B3). This program applies a linear regression analysis to the data points and any p value less than 0.05 is considered significant. Alternatively, if the solvent control is within the 95% confidence limits of the historical mean for control values and the test chemical produces an increase greater than or equal to three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a two-fold increase in the number of histidine-independent revertants.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
Prescreen study:
Results of the prescreen indicated Santowhite ML was not toxic to TA1538 or TA100 at doses of 50.0, 167, 500 and 1670 ug/plate. Inhibited growth (characterized by a reduced background lawn and/or the presence of pindot colonies) was observed at a dose of 5000 ug/plate. The test article precipitated from solution at a dose of 5000 ug/plate.

Mutation assay:
Revertant frequencies for all doses of Santowhite ML in all strains approximated or were less than those observed in the concurrent negative control cultures.

COMPARISON WITH HISTORICAL CONTROL DATA:
All positive and negative control values were within historical limits.

none

Conclusions:

Interpretation of results (migrated information):
negative

Santowhite ML tested in the Salmonella typhimurium reverse mutation assay in concentrations up to 5000 µg/plate did not express any genotoxic potential.

Reference 2

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:

27 October - 5 November, 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to OECD Guideline 471 and under GLP conditions. No relevant deviations reported.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene in S. Typhimurium

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Prescreen test: 0, 10, 33, 67, 100, 333, 667, 1000, 3330, 6670, 10000 µg/plate (TA 100, with and without S9)
Mutation test: 0, 333, 667, 1000, 3330, 6670, 10000 µg/plate (all strains, with and without S9, two independent experiments)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: highest possible dose preparation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: sodium azide, 2-nitrofluorene, 2-amino anthracene, ICR-191

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: one pre-screen test in duplicate, mutation assay in tripicate, two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: counting of revertant colonies and evaluation of bacterial background lawn

Evaluation criteria:

Strains TA98 and TA100:
For a test article to be considered positive, it must cause at least a 2-fold increase in the mean revertants per plate of at least one tester strain over the mean vehicle control value for that tester strain. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.

Strains TA1535, TA1537 and TA1538:
For a test article to be considered positive, it must cause at least a 3-fold increase in the mean revertants per plate of at least one tester strain over the mean vehicle control value for that tester strain. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.

Statistics:

Not mentioned, see evaluation criteria.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: above 333 µg/plate

RANGE-FINDING/SCREENING STUDIES:
Prescreen study:
Dose levels to be tested in the mutagenicity assay were selected based on the results of the dose rangefinding study conducted on the test article using tester strain TA100 in both the presence and absence of S9. Ten dose levels of test article, from 10,000 to 10.0 µg/plate plate were tested. No cytotoxicity was observed in either the presence or absence of S9 as evidenced by no observed decrease in the number of revertants per plate and no thinning of the bacterial background lawn.

Mutation assay:
The data are presented as mean revertants per plate ± standard deviation for each treatment and control group and as individual plate counts. In two independent experiments all data were acceptable and no positive increases in the number of histidine revertants per plate were observed with any of the tester strains either in the presence or absence of S9. All criteria for a valid study were met.

COMPARISON WITH HISTORICAL CONTROL DATA:
All positive and negative control values were within historical limits.

none

Conclusions:

Interpretation of results (migrated information):
negative

The results of the Salmonella/Mammalian-Microsome Reverse Mutation Assay (Ames Test) indicate that in two independent experiments Wingstay L, did not cause a positive increase in the number of histidine revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 February - 15 March, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to OECD Guideline 473 and under GLP conditions. No relevant deviations reported.

Qualifier:

according to guideline

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:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5a medium with 10% FCS, 1% L-glutamine, 1% penicillin and streptomycin

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Dose range finding: 0, 1000 µg/ml (halflog, with and without activation)
Mutation test:
- without activation: 25 - 50 µg/ml in 10 h. assay, 50 - 300 µg/ml in 20 h. assay
- with activation: 100 - 1000 µg/ml in 10 + 20 h. assay

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: and cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION: various incubation and exposure periods in order to optimalize test

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): fluoresence-plus-Giemsa

NUMBER OF CELLS EVALUATED: 100 cells

DETERMINATION OF CYTOTOXICITY:
1. The overall chromosomal aberration frequencies.
2. The percentage of cells with any aberrations.
3. The percentage of cells with more than one aberration.
4. Any evidence for increasing amounts of damage with increasing dose, i.e. a positive dose response.

Evaluation criteria:

1. The overall chromosomal aberration frequencies.
2. The percentage of cells with any aberrations.
3. The percentage of cells with more than one aberration.
4. Any evidence for increasing amounts of damage with increasing dose, i.e. a positive dose response.

Statistics:

Statistical analysis employed the Fisher's Exact Test with an adjustment for multiple comparisons (Sakal and Rohlf. 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls (the solvent and negative controls were statistically evaluated for similarity prior to the pooled evaluation). Test article significance was established where p<0.01. All factors as stated previously were taken into account and the final evaluation of the test article was based upon scientific judgement.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in studies without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed from 101 µg/ml onwards in range-finding study with and without activation

RANGE-FINDING/SCREENING STUDIES:
Range-finding:
Total cellular toxicity was observed in the culture dosed with 1010 µg/ml, severe cell cycle delay was evident in the cultures dosed with 101 and 337 µg/ml in the rangefinding assay without metabolic activation. Severe reduction in the mitotic index was observed in the cultures dosed with 997, 332and 99.7 µg/ml in this assay.
No cell cycle delay or a significant reduction in mitotic index was evident in the cultures analyzed in the rangefinding assay with metabolic activation.

Mutation assay:
Cultures of CHO cells were incubated with target concentrations of 25.0 to 50.0 µg/ml of the test article solution in a 10 hour aberrations assay and with 50.0 to 300 µg/ml in a 20 hour aberrations assay under nonactivation conditions.Target concentrations of 100 to 1000 µg/ml were tested in 10 and 20 hour aberrations assays with metabolic activation. These results were verified in independently conducted confirmatory trials. No significant increase in cells with chromosomal aberrations was observed at the concentrations analyzed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Range-finding assay without activation: total cellular toxicity was observed in the culture dosed with 1010 µg/ml, severe cell cycle delay was evident in the cultures dosed with 101 and 337 µg/ml.
Range-finding assay with activation: minor cellular toxicity was observed in the culture dosed with 1010 µg/ml.
Mutation assay without activation: cellular toxicity was observed in cultures dosed 300 µg/ml onwards.
Mutation assay with activation: minor cellular toxicity was observed in the culture dosed with 1000 µg/ml.

none

Conclusions:

Interpretation of results (migrated information):
negative

The results of the chromosomal aberration assay in CHO cells indicate that Wingstay L was negative for inducing chromosomal aberrations in experiments with and without S9-activation. Cellular toxicity and a reduction in the mitotic index was observed in experiments without S9-activation.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

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: The study report lacks information such as followed guidelines and quality control of the tested substance, which is needed for drawing firm conclusions.

Qualifier:

according to guideline

Guideline:

other: Health, Safety and Government Compliance test method 79-11

GLP compliance:

not specified

Target gene:

E. Coli Pol. A1

Species / strain / cell type:

E. coli, other: W3110 and 3478

Metabolic activation:

with and without

Test concentrations with justification for top dose:

0, 10, 100, 1000 µg/plate

Non-activation test results:

The test chemical Wingstay L, did not induce preferential inhibition of the Pol Al- strain as compared to the solvent control and to the Pol A+ strain. The limit of chemical solubility in the test medium was 10 mg/ml. Neither a dose-response relationship nor a toxic response to the chemical was observed. The final survival indices were obtained by calculating a weighted average of data from chronologically independent tests.

Activation test results:

No preferential inhibition of the Pol Al- strain as compared to the solvent control and to the Pol A+ strain was observed with the addition of the metabolic activation system. All test criteria for a negative response were satisfied. The final survival indices were obtained by calculating a weighted average of data from chronologically independent tests.

Conclusions:

Interpretation of results (migrated information):
negative

Wingstay L may be considered negative in the E. Coli Pol A1- assay for DNA damage, although not fully reliable.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 February - 20 May, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to OECD Guideline 476 and under GLP conditions. No relevant deviations reported.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium with 8% FBS, L-glutamine, penicillin and streptomycin

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Dose range finding: 0, 1000 µg/ml (3-log, with and without activation)
Mutation test: 100 - 1000 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

Migrated to IUCLID6: and 5-bromo-2-deoxyuridine (BrdU)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 16 - 18 h.
- Exposure duration: 4 h.
- Expression time (cells in growth medium): 6 days

NUMBER OF CELLS EVALUATED: at least 50 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and growth rate

Evaluation criteria:

A dose-related or toxicity-related increase in mutant frequency should be observed. It is desirable to obtain this relation for at least three doses. However, this depends on the concentration steps chosen for the assay and the toxicity at which mutagenic activity appears.

If an increase in mutant frequency is observed for a single dose near the highest testable toxicity, as defined previously, and the number of mutant colonies is more than twice the value needed to indicate a significant response, the test article generally will be considered mutagenic. Smaller increases at a single dose near the highest testable toxicity will require confirmation by a repeat assay.

Statistics:

The statistical tables provided by Kastenbaum and Bowman (1970) are used to determine whether the results at each dose level are significantly different from the negative controls at 95% or 99% confidence levels. This test compares variables distributed according to Poissonian expectations by summing up the probabilities in the tails of two binomial distributions. The 95% confidence level must be met as one criterion for considering the test article to be active at a particular dose level. In addition, the mutant frequency must meet or exceed 15 x 10 6 in order to compensate for random fluctuations in the 0 to 10 x 10 6 background mutant frequencies that are typical for this assay.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed from 31.1 µg/ml onwards

RANGE-FINDING/SCREENING STUDIES:
Range-finding:
Study without activation: weak toxic at 1000 µg/ml. Study with activation: moderate toxic at 500 and 1000 µg/ml.

Mutation assay:
Study without activation: dose-related toxicities observed in two independent experiments. The mutant frequency was within the background range.
Study with activation: dose-related toxicities observed in two independent experiments. The mutant frequency was within the background range.

none

Conclusions:

Interpretation of results (migrated information):
negative

Wingstay L is considered negative for inducing forward mutations at the HGPRT locus in Chinese hamster ovary cells under both the S9 metabolic activation and nonactivation conditions of the assay. Cellular toxicity was observed in experiments with and without S9-activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the results of the genotoxicity studies, the substance does not need to be classified according to the CLP Regulation.