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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

4,4`-sulfonyldiphenol is negative in different Ames tests and a gene mutation test in cultured mammalian cells (HGPRT assay). Positive effects were observed in two in vitro chromosomal aberration tests in cultured CHO cells in the absence of metabolic activation only. These effects were not reprodicable in a GLP study according to current guideline.

Genetic toxicity in vivo

Description of key information

In an in vivo mouse micronucleus assay 4,4`-sulphonyldiphenol does not induce cytogenetic damage in bone marrow cells of NMRI mice and is thus not considered to be mutagenic in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-03-26 to 2010-05-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: 28.03 g (mean body weight)
- Housing: Makrolon cages, type M II; single housing
- Diet (e.g. ad libitum): Type Lignocel PS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany
- Water (e.g. ad libitum): Drinking water from bottles was available ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 - 18.00 hours; 12 hours darkness from 18.00 - 6.00 hours
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Type and concentration of dispersant aid (if powder): DMSO is used up to 4 mL/kg body weight only
Duration of treatment / exposure:
The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle. The positive controls, both dissolved in purified water were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a volume of 10 mL/kg body weight. The animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48 hours (highest test substance concentration, vehicle) after the treatment, respectively.
Frequency of treatment:
24, 48 hours after dose administration
Dose / conc.:
500 other: mg/L bw (nominal)
Dose / conc.:
1 000 other: mg/L bw (nominal)
Dose / conc.:
2 000 other: mg/L bw (nominal)
No. of animals per sex per dose:
5 male animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide (CPP); vincristine sulfate (VCR)
- Doses / concentrations: 20 mg/kg bw (CPP); 0.15 mg/kg bw (VCR)
Tissues and cell types examined:
The micronucleus study evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male mice.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, at 2 000 mg/kg body weight recommended as the highest dose according to the OECD Guideline all animals (male and female) survived. Weak signs of toxicity (piloerection) were observed over two days. However, there were no distinct differences in the clinical observations between males and females. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 2 000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1 000 mg/kg and 500 mg/kg body weight were administered as further doses.

METHOD OF ANALYSIS:
For the determination of the test substance concentrations in the vehicle, 6 samples of each dose were taken from the test substance preparations. These were kept at room temperature until the treatment of the last animal (approximately 1 hour) and then were kept deep-frozen. The determination of the concentrations in the vehicle was carried out by means of HPLC The homogeneity of the samples and the stability of the test substance in the vehicle mixture DMSO/corn oil (ratio 2:3) were confirmed indirectly based on these data.
Evaluation criteria:
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs.
- The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN. The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No dose-dependent inhibition of erythropoiesis induced by the treatment of mice with 4,4`-sulphonyldiphenol was detected. The ratio of polychromatic to normochromatic erythrocytes was in the range of the vehicle control values in all dose groups.
The administration of the test substance led to weak clinical signs of toxicity.

No other information

Conclusions:
Interpretation of results: negative
The test substance 4,4`-sulphonyldiphenol has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.
Executive summary:

4,4`-sulphonyldiphenol was tested in a mouse micronucleus assay according to OECD guideline 474 and EU-method B.12. The test substance, dissolved in DMSO and emulsified in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the single oral administration of 4,4`-sulphonyldiphenol did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was within the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data. Thus, under the experimental conditions of this study, the test substance 4,4`-sulphonyldiphenol does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo. The administration of the test substance led to weak clinical signs of toxicity.

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

Additional information

In-vitro studies: Bacterial systems

An Ames test using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 at concentrations ranging from 0.16-5000 µg/plate with and without metabolic activation was performed according to the current guideline study under GLP (ICI Chemicals and Polymers, 1989). 4,4`-sulfonyldiphenol did not induce any significant increases in the observed numbers of revertant colonies even at bacteriotoxic test concentrations.

In supporting study (CIBA-Geigy, 1987), 4,4`-sulfonyldiphenol was tested in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at five dose levels: 20, 80, 320, 1280 and 5120 pg per Petri dish. Every concentration was tested in triplicate.

4,4`-sulphonydiphenol was not mutagenic for S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100.

Similar results were observed in further Ames tests in which concentrations ranging from 20-5000 µg/plate showed no mutagenic effect when tested in 5 strains (TA98, TA100, TA1535, TA1537 and WP2uvrA) with and without metabolic activation (JETOC, 1996 and OECSEH, 1999). These results were further confirmed in 2 other tests using 4 strains (Seifried, 2006 and Bayer, 1991).

Combining all in-vitro studies in bacterial system, 4,4`-sulfonyldiphenol was negative in the Ames test even if tested at clear bacteriotoxic concentrations.

In-vitro studies: Mammalian cell gene mutation test

4,4`-sulfonyldiphenol was tested for cell gene mutation in the HGPRT assay in CHO cells at dose levels up to 1000 µg/ml with and without metabolic activation system (Amoco, 1990). There were no significant increases in the mutant frequencies in any of the test groups. Under the conditions of this assay, 4,4`-sulfonyldiphenol was negative in the CHO/HGPRT mutation assay both with and without S9 mix even if tested at cytotoxic concentrations.

In-vitro studies: Chromosome aberration tests

In a GLP-compliant guideline study the test substance was assessed for its potential to induce structural chromosome aberrations and / or changes in the number of chromosomes in V79 cells in vitro. Three independent experiments were carried out, both with and / or without the addition of liver S9 mix from induced rats (exogeneous metabolic activation). Cells were exposed for 4 and 18 hours with sampling times of 18 and 28 hours at 0 - 1000 µg/mL in the absence and presence of S9 mix and in the absence of S9 mix exposed for 18 hours with a sampling time of 18 hours at 0 - 400 µg/mL. Cytotoxicity indicated by clearly reduced relative population doubling (RPD) or mitotic rates was observed at least at the highest applied test substance concentration in all experimental parts of the study. On the basis of the results the test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S9 mix or after adding a metabolizing system. The observation of an increase in the frequency of cells containing numerical chromosome aberrations in the 1st Experiment in the absence of S9 mix was considered as artificial. Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging potential under in vitro conditions in V79 cells in the absence and presence of metabolic activation (BASF 2017).

In a chromosomal aberration assay in Chinese hamster Ovary (CHO) cells 4,4`-sulfonyldiphenol was tested at dose levels of 125-1000 µg/ml with and without metabolic activation system (Amoco, 1991). A statistically significant (p≤ 0.01) increase in chromosome aberrations was observed at dose levels of 500 and 600 µg/ml in the absence of a metabolic activation system. At these dose levels mild cytotoxicity was observed (25 -35%). This cytotoxic effect of 4,4'-dihydroxydiphenyl sulphone precluded the analysis of the higher test concentrations in the assay.

Reporting of a further guideline study using Chinese Hamster Ovary (CHL) cells is of limited quality. Cells were treated for 24 and 48h at 0 -0.4 mg/ml in the absence of S9 mix as the continuous treatment and for 6h at 0-1.4 mg/ml in the absence and presence of S9 mix as the short term treatment. In the 24-h continuous treatment, structural chromosome aberration was significantly increased at the high dose concentration (0.40mg/mL) and the incidence was 5.5% (including gaps). Polyploid cells were not increased in the continuous treatments. Although number of polyploidy was significantly increased in the short-term treatments with or without S9, it was judged to be negative since the frequency of induction was low. Cytotoxicity was observed at 0.70-1.4 mg/mL in the short-term treatment with and without S9 mix. Desribed observations are not reproducible and the assay can not be used for the assessment of in vitro genotoxicity in mammalian cells (OECSEH, 1999). The results were judged to be of no biological significance because of low frequency.

Retrospective evaluation of two databases, where test results were obtained under OECD-GLP or compliant Japanese test guideline under GLP and measured RCC data were available has been conducted by Fujita et al. (2016). All chemicals in the databases were assessed using Chinese hamster lung (CHL/IU) cells under short-term treatment (with and without metabolic activation) and / or continuous treatment (24 h, without metabolic activation). First, the lowest dose at which a positive response (chromosomal aberration rate of 5 % or more) was observes was defined as the LOGEL, whereas the dose one step lower was defined as the NOGEL. Next, the measured RCC (mRCC) at the LOGEL/NOGEL was designated as the mRCC(LOGEL/NOGEL). Similarly, the estimated RICC (eRICC) obtained using the formula RICC = 1.5 * RCC - 0.49 was designated as the eRICC(LOGEL/NOGEL). The test substance was evaluated as probably negative chemical in the retrospective evaluation of several chemicals by applying a mathematical approach to estimate new incidences from the relative cell count (RCC).

In-vitro studies: Cell free assay (Microtubule polymerization)

4,4`-sulfonyldiphenol was tested for its potential to interfere with the formation of microtubule (MT) in a cell-free assay (Pfeiffer et al. 1996). This MT polymerization assay was carried out with MT proteins from bovine brain at test concentrations of 50-200 µM. The test substance did not inhibit MT assembly a potential indication for aneuploidic effects.

In-vivo studies: Mouse micronucleus assay

4,4`-sulphonyldiphenol was tested in an in vivo mouse micronucleus assay according to OECD guideline 474 and EU-method B.12 (BASF, 2010). The test substance, dissolved in DMSO and emulsified in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. According to the results of the present study, the single oral administration of 4,4`-sulphonyldiphenol did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was within the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data. Thus, under the experimental conditions of this study, the test substance 4,4`-sulphonyldiphenol does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo. The administration of the test substance led to weak clinical signs of toxicity.

In the final decision on substance evaluation pursuant to article 46(1) of Regulation (EC) No 1907/2006 for 4,4'-sulfonyldiphenol (CAS 80-09-1; EC 201-250-5) ECHA has stated that the Registrants are requested to carry out anIn vivoalkaline comet assay performed in rats by oral administration (gavage) (test method: OECD 489) on tissues (at least the liver, glandular stomach and duodenum).

Based on the comments of the Registrants during the commenting phase ECHA agrees that data requirements can also be fulfilled by a tiered approach. In the final decision ECHA has stated that the in vivo comet assay shall not be performed if both of the following conditions are fulfilled:

1. The results of the toxicokinetics study demonstrate the presence of the parent substance in the plasma, and

2. It is demonstrated that the results of this in vivo toxicokinetics study in rats can be used to confirm the results of the available in vivo micronucleus study in mice. A justification shall be provided for the ability to use the rat data and any other available data to confirm the results of the mouse study and to demonstrate that the conditions specified in the OECD guidance 474 (adopted in 26 September 2014) regarding the target tissue exposure (paragraph 40) are fulfilled.

 

The Registrants decided to follow the tiered approach:

The two availablein vitromammalian chromosomal aberration assays were not performed in accordance to the current testing guideline, e.g. significantly prolonged treatment periods were applied. In both assays a statistically significant induction of chromosomal aberrations for 4,4`-sulphonyldiphenol is only described in the highest tested concentrations, in presence of significant cytotoxicity and in absence of metabolic activation. A dose response as requested by the OEDC TG 473 could not be established and in conclusion a high dose effect cannot be excluded. In the assay performed in Chinese Hamster Ovary (CHO) cells (Amoco 1991) a statistically significant increase in cells with aberrations was only observed in the highest tested dose (0.5 mg/ml) in absence of metabolic activation, though in presence of significant cytotoxicity as demonstrated by a 34-36% decrease in mitotic index as an insensitive parameter of cytotoxicity. Exposure time was 13h instead of 3-6h required by the guideline. The assay performed with Chinese Hamster Lung (CHL) cells (OECSEH 1999) did not result in a statistically significant increase in cells with aberrations with a 6h treatment period as requested by the guideline. A weak (4.5%) increase in cells with aberrations was only observed with the highest tested concentration (0.4 mg/ml) and a 24h treatment period in absence of metabolic activation. At this dose level significant cytotoxicity was identified, demonstrated by only 37.5% cell confluency in comparison to the control group. A confirmatory assay was not performed. In a re-evaluation of the assay the “…CAs observed are not considered to be of biological significance…” (Morita et al. 2012).

A retrospective evaluation of two databases, where test results were obtained under OECD-GLP or compliant Japanese test guideline under GLP and measured RCC data were available has been conducted by Fujita et al. (2016). All chemicals in the databases were assessed using Chinese hamster lung (CHL/IU) cells under short-term treatment (with and without metabolic activation) and / or continuous treatment (24 h, without metabolic activation). First, the lowest dose at which a positive response (chromosomal aberration rate of 5 % or more) was observes was defined as the LOGEL, whereas the dose one step lower was defined as the NOGEL. Next, the measured RCC (mRCC) at the LOGEL/NOGEL was designated as the mRCC(LOGEL/NOGEL). Similarly, the estimated RICC (eRICC) obtained using the formula RICC = 1.5 * RCC - 0.49 was designated as the eRICC(LOGEL/NOGEL). 4,4`-sulphonyldiphenol was evaluated as probably negative chemical in the retrospective evaluation of several chemicals by applying a mathematical approach to estimate new incidences from the relative cell count (RCC).

In addition, a newin vitromammalian chromosomal aberration assay in V79 cells according to guideline (OECD 473) and GLP was performed to investigate if the positive results of the above-mentioned studies could be confirmed (BASF 2017). Three independent experiments were carried out, both with and / or without the addition of liver S9 mix from induced rats (exogeneous metabolic activation). Cells were exposed for 4 and 18 hours with sampling times of 18 and 28 hours at 0 - 1000 µg/mL in the absence and presence of S9 mix and in the absence of S9 mix exposed for 18 hours with a sampling time of 18 hours at 0 - 400 µg/mL. Cytotoxicity indicated by clearly reduced relative population doubling (RPD) or mitotic rates was observed at least at the highest applied test substance concentration in all experimental parts of the study. On the basis of the results the test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S9 mix or after adding a metabolizing system. The observation of an increase in the frequency of cells containing numerical chromosome aberrations in the 1st Experiment in the absence of S9 mix was considered as artificial. Thus, under the experimental conditions described, the test substance 4,4`-sulphonyldiphenol was considered not to have a chromosome-damaging potential under in vitro conditions in V79 cells in the absence and presence of metabolic activation.

4,4`-sulphonyldiphenol was also tested in an in vivo mouse micronucleus assay according to OECD guideline 474 and EU-method B.12 (BASF, 2010). The test substance, dissolved in DMSO and emulsified in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. According to the results of the present study, the single oral administration of 4,4`-sulphonyldiphenol did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was within the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data. Thus, under the experimental conditions of this study, the test substance 4,4`-sulphonyldiphenol does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo. The administration of the test substance led to weak clinical signs of toxicity. To confirm the systemic availability of the substance and by that the relevance of the negative result of this in vivo study a toxicokinetics study is currently ongoing to demonstrate the presence of the parent substance in the plasma. As soon as these results will be available a final conclusion on the hazard identification including classification and labelling for the endpoint genotoxicity will be made.

Justification for classification or non-classification

Based on the available results of the three different in vitro and one vivo genetic toxicity studies, 4,4`-sulphonyldiphenyl is considered to be non genotoxic in vivo and accordingly has not to be classified and labelled as genotoxic according to Directive 67/548/EEC (DSD) and Regulation No 1272/2008 (CLP).