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Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assays with pitch, coal tar, high-temp. (CTPht) or surrogates are positive. Other genetic toxicity tests in vitro with CTPht could not be located. But additional studies are not necessary, because CTPht is classified as carcinogenic and mutagenic according to Regulation (EC) No 1272/2008 (including amendments), Annex VI, Part 3, Table 3.1 (classification Carc. 1A and Muta 1B).

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Jul. - 03 Sep. 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
Plate incorporation test
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): pitch (coal tar)
- Lot/batch No.: ATE no. 10444
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Microsomal fraction prepared from induced livers of male Wistar rats, induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) orally (3x)
Test concentrations with justification for top dose:
1st experiment: 3.16, 10, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (TA 98, TA 100: +/-S9)
31.6, 100, 316, 1000, 2500, and 5000 µg/plate (TA 1535: +/-S9)
10, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (TA 1537, TA 102: +/-S9)
2nd experiment: 1.58, 5.0, 15.8, 50, 158, 500, 1580, and 5000 µg/plate (TA 98, TA 1537: +/- S9)
15.8, 50, 158, 500, 1580, and 5000 µg/plate (TA 100, TA 1535, TA 102: +/- S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with survival of bacteria and S9 activity
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: see Report p. 16
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth/colony formation

Evaluation criteria:
Considered as mutagenic
- if a clear and dose-related increase in the number of revertants occurs in at least one tester with or without metabolic activation
and/or
- if a biologically relevant positive response for at least one of the dose groups occurs in at least one tester with or without metabolic activation.

An increase is considered relevant
- if in TA 100 and TA 102 mutation rate is at least twice as high as the rate of the solvent control;
- if in TA 98, TA 1535, and TA 1537 the mutation rate is at least 3x higher than that of the solvent control.
Statistics:
According to the OECD guidelines, the biological relevance is the criterion for the interpretation of the results: a statistical evaluation was not considered necessary under this premise (report p. 22).
Key result
Species / strain:
S. typhimurium, other: TA 100, TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
reproducible, dose response
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
reproducible, dose response
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, at test concentrations of >= 158 µg/plate


No biologically relevant increases in TA 1535 and TA 102.

Biologically relevant increases in TA 98 (-S9, reproducible) ==> Mutation factor =< 6.6

Biologically relevant increases in TA 98, TA 100, and TA 1537 (+S9) ==> Mutation factor =<24.6 in TA 98

Conclusions:
The results of the bacterial reverse mutation test show a positive mutagenic response of pitch, coal tar, high-temp. in three out of five Salmonella typhimurium tester strains, in one with and without and in two only with metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): BX90/2563 (electrode binder)
- Physical state: solid, black powder
- Treatment of test material prior to testing: 2 g test material powder was stirred in a closed bottle protected from light for 24 h with 150 ml of the solvent. The insoluble particles were removed by filtration. The solution contained 12.4 mg/mL (approx. 93 % of nominal loading).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Arochlor-induced liver microsomes (Wister rats)
Test concentrations with justification for top dose:
First main test (100 µL/plate): 77.5, 155, 310, 620, 1240 µg/plate (mass related to dissolved pitch)
Second main test (50 µL/plate): 1, 5, 24.8, 124, 120 µg/plate
Repetition test same as second main test
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: high dissolution potential for the test material combined with relatively low cytotoxicity to the tester strains
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-Nitro-1,2-phenylene diamine: TA 98 (without metabolic activation); 2-Aminoanthracene: all strains (with metabolic activation))
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium, in agar (plate incorporation)
Evaluation criteria:
A reproducible and dose-related increase in at aleast one strain is considered positive, 
- for TA98 and 1537: a 2-fold increase vs. control
- for TA1535: a 3-fold increase vs. control
- for TA100: a 1.5-fold increase vs. control
Statistics:
Kruskal-Wallis U-test applied in the second test series to confirm positive results.
Key result
Species / strain:
S. typhimurium, other: TA 100, TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
reproducible, dose-related
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
conc. 1240 µg/plate (TA 100)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
reproducible, dose-related
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: following dosing of 100 and 50 µL solution containing >= 124 µg

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity of the solvent that was diminished by reducing the dosing volume to 50 µL per plate
from 100 to 50 µL per plate (Report, p. 17)

The results may be categorised as follows:

-S9                     +S9

==================================

TA 15                     -                   -

TA 1537                 -                +++ MF = <56

TA 98                     +               +++ MF = <33

TA 100                  -/+                  + MF = <5

==================================

- = no mutagenic activity

-/+ = not to slightly enhanced

+ = weak but significant

++ = moderate

+++ = strong

MF = mutation factor above solvent control

-----------------------------------------------------

Conclusions:
The results of the bacterial reverse mutation test show a positive mutagenic response of pitch, coal tar, high-temp. in three out of four Salmonella typhimurium tester strains, in one with and without and in two only with metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, reduced test design for comparative purposes, including detailed analysis, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only TA 98 used; S9 prepared from hamster liver
Principles of method if other than guideline:
Comparative study including various asphalts and pitches, combined with high analytical efforts
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Source of test material: fume condensates of coal tar pitch representing ASTM type I specifications for roofing products generated at a temperature of 232 °C and 316 °C, respectively
- Composition: fume condensates contained benzo[a]pyrene together with other PAH (three- up to seven-rings)
- Concentration of PAH in fume condensates: fumes generated at 316 °C contained significantly higher concentrations of PAH than those generated at 232 °C (for details see Reference, Tab. 2)
- Treatment of test material prior to testing: test solutions were prepared by agitating fume condensates in DMSO at 200 mg/mL at a tempreature of 60 °C for 1 h. The resulting material was cleared from solid matter by centrifugation.
- Concentration of BaP in source coal tar pitch: 1.81 %
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
with
Metabolic activation system:
from Arochlor-induced hamster liver (Microbiol. Associates)
Test concentrations with justification for top dose:
no data
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: increase in sensitivity of the test response (modification according to Blackburn et al. 1984, 1986)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
NBS No. 1618: residual fuel oil
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 h on plate at 37 °C
Statistics:
Nonlinear regression after Myers et al. 1981 for dose-response relationship
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

There was a good correlation between the B[a]P content and mutagenic activity (Report Fig 1). Coal tar pitch fumes generated at 316 °C contained significantly higher concentrations of PAH than those generated at 232 °C and the mutagenic activity paralleled the PAH content.

Conclusions:
Fume condensates of coal tar pitch displayed a positive mutagenic response in tester strain TA 98 in the bacterial reverse mutation assay (Ames test). In this study, only one strain of S. typhimurium was tested.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material are fume condensates of coal tar pitch (ASTM type 1 specifications for roofing products). These condensates contain mainly PAH (three up to seven rings). Biologically active components in the target material pitch, coal tar, high-temp. are also PAH (mostly four to six rings). As the active ingredients in both materials are the same, data resulting from the source material can be used for characterising the toxic properties of the target substance CTPht.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source test material are fume condensates of coal tar pitch (ASTM type 1 specifications for roofing products). Fumes condensates are generated at 232 °C and 316 °C resulting in liquid mixtures containing mainly PAH (three- up to seven-ring PAH). PAH concentrations are higher in the condensate obtained at higher temperature.
The target material pitch, coal tar, high-temp. is the product (residue) of the oxygen free high-temperature distillation of coal tars and consists of a complex mixture of polycyclic aromatic hydrocarbons, predominantly of highly condensed aromatic ring systems forming an inert matrix. In this matrix, individual PAH with a lower degree of condensation are included. They consist mostly of 4- and 5-membered (up to 6) ring aromatic PAH. The material is solid and only a fraction of ca. 20 % will evaporate at a distillation temperature up to 500 °C. Even if the basic structure of both materials, source and target material, is quite different, the components that will act on biological samples are similar.

3. ANALOGUE APPROACH JUSTIFICATION
Due to the liquid state of the fume condensates, their constituents are directly available under ambient and environmental conditions. These are three- to seven-ring PAH. From use under environmental conditions or during processing of CTPht, PAH (four-, mainly five-, up to six-ring representatives) included in the solid material may be released and will also be biologically available. After contact with/intake by biological organisms, PAH can exert there adverse mode of action. Adverse effects observed with the source material (fume condensates) are assumed to be more severe as with the target material, because PAH from the source material are freely available, but release of PAH from the target material CTPht is much more limited. But overall, the active components are mostly the same.
For these reasons, it is considered justified to use genotoxicity data of fume condensates from coal tar pitch in order to characterise effects of pitch, coal tar, high-temp. Using data from fume condensates of coal tar pitch may even result in overestimation of risk for CTPht.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to preceding entry:
Source test material: coal tar pitch fume condensate;
Reference: Machado ML et al. 1993
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: the result of the test substance is adopted for the target substance CTPht
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

For pitch, coal tar, high-temp., no information on genetic toxicity in vivo is available. Additional information is not required, because the substance is classified as Carc. 1A and Muta. 1B according to Regulation (EC) No 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data from Ames-tests only have been provided, since coal-tar pitch is classified as mutagenic and carcinogenic. In one of the tests, coal tar pitch (fume condensate) was used as supporting substance.

Justification for classification or non-classification

Pitch, coal tar, high-temp. contains benzo[a]pyrene in concentrations up to 1.5 %. BaP is known to be mutagenic and is classified as mutagenic Cat. 1B according to Regulation (EC) No 1272/2008 (CLP regulation). In Ames tests, pitch, coal tar, high-temp. was demonstrated to be mutagenic. Based on results of the bacterial reverse mutation assays and of its BaP content, pitch, coal tar, high-temp. is self-classified by the registrant as mutagenic Cat. 1B in compliance with Regulation 1272/2008. According to CLP regulation Annex VI, Part 3, Table 3.1 amended by Commission regulation (EU) No 944/2013, pitch, coal tar, high-temp. is classified as Muta. 1B.