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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
There are 2 key endpoints required for in vitro genetic toxicity: Ames test - The test material was tested in the histidine-deficient strains of Salmonella typhimurium TA98, TA100, and TA102 and in the tryptophan-deficient strain of Escherichia coli WP2 uvrA at dose levels of 0.033 to 3.33 mg/plate with and without metabolic activation provided by Aroclor-induced rat liver S-9. The test material was suspended in 25% Pluronic 127 (w/w in ethanol). The suspension was miscible with the top agar. It was not cytotoxic at any concentration tested. Under the conditions tested, the test material was not mutagenic to TA98, TA100, TA102, or E. coli WP2 uvrA. Mouse lymphoma - The test material was tested in the L5178Y TK+/- Mutagenicity Screen with and without S-9 metabolic activation. The cultures with activation were tested at concentrations ranging from 75 µg/ml to 275 µg/ml; cultures without activation were tested at concentrations ranging from 60 µg/ml to 110 µg/ml. The results indicated that the test material did not induce a significant increase in the mutant frequencies of cultures tested either with or without metabolic activation. Under the conditions tested the test material was not mutagenic in this screen. In vivo: Based on the results of the dose selection study, the maximum tolerated dose was estimated as >5000 mg/kg. The test article did not induce a statistically significant increase in micro-nuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse micronucleus assay.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
11th September to 11th October 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed per recognized guideline following GLP. This study is being used as read across from Phenol, tetrapropenyl-, sulfurized, calcium salts CAS No. 68855-45-8, therefore reliability is reduced to 2.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Crl:CD-1®(ICR) BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI.
- Age at study initiation: All animals were eight weeks and three days old at the time of dosing.
- Weight at study initiation: The weight range of the animals used in the micronucleus assay was 23.7-35.1 and 21.2-28.2 grams for the males and females, respectively.
- Assigned to test groups randomly: Yes
- Housing: Animals were housed five per cage during quarantine, and housed at least five per cage at randomization. Sanitized caging was used for housing the animals.
- Diet/water (e.g. ad libitum): A commercial diet (Purina® Certified Laboratory Pellets ® # 5002) and water were available ad libitum for the duration of the study. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analyzed on a retrospective basis for specified microorganisms, pesticides, alkalinity, heavy metals, and halogens.
- Acclimation period: Animals were quarantined for seven days before being placed on study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 6 °F
- Humidity (%): 55 ± 15%
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained

Route of administration:
intraperitoneal
Vehicle:
The vehicle control, peanut oil (Sigma, Lot # 83H0848), was administered concurrently with the test article at a volume of 10 mL/kg.
Details on exposure:
Dosing suspensions were prepared just prior to dosing and were prepared by making a 500 mg/mL stock for the high dose (5000 mg/kg ). This was prepared by adding peanut oil (Sigma, Lot # 83H0848) to the test material resulting in a dark brown viscous solution. Dilutions of this stock were prepared for the 2500 and 1250 mg/kg and dose levels. Volumes dosed were 10 mL/kg and were based upon individual animal weights. All dosing stocks were continuously mixed during the dosing procedure.
Duration of treatment / exposure:
The animals dosed with the test article and the vehicle control were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow.
Frequency of treatment:
Once intraperitoneally
Post exposure period:
24, 48 and 72 hours post-dose
Remarks:
Doses / Concentrations:
0, 1250, 2500 and 5000 mg/kg b.wt.
Basis:
nominal conc.
The dose volume was 10 mL/kg b.wt. for all groups.
No. of animals per sex per dose:
Five mice/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide at a dose of 60 mg/kg b.wt.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on results from the dose selection study, dose levels of 1250, 2500 and 5000 mg/kg were selected for testing in this study.

DETAILS OF SLIDE PREPARATION:
At the appropriate harvest time, the animals were euthanized by CO2/O2 inhalation followed by penetration of the thorax. A limb bone was removed from each hind leg and the adhering soft tissue and epiphyses were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3 - 5 mL bovine serum (one tube for each animal). Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, and stained in May-Grunwald solution followed by Giemsa (Schmid, 1975). The air-dried slides were coverslipped.

METHOD OF ANALYSIS:
The slides were coded for analysis, and scored for micronuclei and the polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this Crl:CD-1®(ICR) BR strain is about 0.0 - 0.4%.

The frequency of PCEs versus NCEs was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring at least the first 1000 erythrocytes.

Evaluation criteria:
The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ringshaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).
Statistics:
Results for each sex / harvest time were analyzed by ANOVA on either untransformed (when variance homogeneous) or rank transformed (when
variance heterogeneous) micronucleus cell count data. If significance was observed with ANOVA, a Dunnett’s t-test was used to determine which
dose groups were different from the negative control. A Cochran-Armitage test for linear trend was used to evaluate dose-response.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Dose levels of 1625, 2750, 3875 and 5000 mg/kg were administered by intraperitoneal injection for the dose selection study.
- Clinical signs of toxicity in test animals:
All animals were examined after dosing and daily throughout the duration of the study (three days) for toxic effects and/or mortalities. All animals appeared normal immediately after dosing and remained healthy until the end of the observation period. No mortality occured.
- Rationale for exposure: Based on these results, the maximum tolerated dose was estimated to be >5000 mg/kg.


RESULTS OF DEFINITIVE STUDY
All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest times. All test article dosed groups appeared normal immediately after dosing and remained healthy until the appropriate harvest times.
The test article induced no statistically significant increases in micro-nucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The test article did not induce a statistically significant change in the PCE:NCE ratio. The positive control, Cyclophosphamide, induced statistically significant increases in micronucleated PCEs in both sexes as compared to the vehicle controls, with means and standard errors of 2.38% ± 0.54% and 4.28% ± 0.79% for the males and females, respectively.

There was no mortality and all animals appeared normal without sign of adverse effect until sacrifice. Mean percentage of micronucleated PCEs was within the range of laboratory historical controls for all treatment and vehicle control groups.

Conclusions:
Interpretation of results (migrated information): negative
The test article did not induce a statistically significant increase in micro-nuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse micronucleus assay.

Executive summary:

In a study conducted in accordance with GLP to OECDguideline 474, based on the results of the dose selection study, the maximum tolerated dose was estimated as >5000 mg/kg. In the micronucleus assay, the test article was suspended in peanut oil and dosed by intraperitoneal injection at 1250, 2500 and 5000 mg/kg. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. The animals dosed with the test article and the vehicle control were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. The animals dosed with the positive control were euthanized approximately 24 hours after dosing for extraction of the bone marrow.

One thousand PCEs per animal were scored. The test article did not induce a statistically significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test. The test article did not induce a statistically significant change in the PCE:NCE ratio.

The test article did not induce a statistically significant increase in micro-nuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse micronucleus assay.

There was no available data to fulfil this endpoint for the test material and so the study was read-across from a supporting substance (Phenol, tetrapropenyl-, sulfurized, calcium salts CAS No. 68855-45-8).

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

Additional information

Additional information from genetic toxicity in vivo:

The discussion includes a summary of the data as well as additional genotoxicity modeling conducted to demonstrate that read across is appropriate and genotoxicity is not expected.

In vitro studies:

Ames test - In this key study for in vitro genetic toxicity (Machadoet al, 1985, report number: SOCAL 2321) there was no guideline specified, however it was considered to be comparable to OECD Guideline 471 (Bacterial Reverse Mutation Assay). The study was conducted in line with GLP. A reliability rating of 1 according to the criteria of Klimisch, 1997.

Two additional Ames tests are included (Lawlor, 1997 and Machado et al., 1986) for surrogate test materials to cover additional strains not tested in the key study (Machado et al., 1985).

Mouse lymphoma- In this key study for in vitro genetic toxicity (Winingeret al, 1985, report number: SOCAL 2322) there was no guideline specified, however it was considered to be comparable to OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test). The study was conducted in line with GLP. A reliability rating of 1 according to the criteria of Klimisch, 1997.

In vivo study:

In the key study for in vivo genetic toxicity (Ivett, 1997, Corning Hazleton report number: 17865-0-455CO) the study was conducted according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test). The study was conducted in line with GLP.

The reliability rating for this study is 1, however this is being used as read across from a supporting substance (Phenol, tetrapropenyl-, sulfurized, calcium salts CAS No. 68855-45-8) as there was no available data to fulfil this endpoint for the test material and so the reliability rating will be reduced to 2, according to the criteria of Klimisch, 1997.

The following information is taken into account for any hazard / risk assessment:

There are 2 key endpoints required for in vitro genetic toxicity:

Ames test - The test material was tested in the histidine-deficient strains of Salmonella typhimurium TA98, TA100, and TA102 and in the tryptophan-deficient strain of Escherichia coli WP2 uvrA at dose levels of 0.033 to 3.33 mg/plate with and without metabolic activation provided by Aroclor-induced rat liver S-9. The test material was suspended in 25% Pluronic 127 (w/w in ethanol). The suspension was miscible with the top agar. It was not cytotoxic at any concentration tested.

Under the conditions tested, the test material was not mutagenic to TA98, TA100, TA102, or E. coli WP2 uvrA.

Mouse lymphoma - The test material was tested in the L5178Y TK+/- Mutagenicity Screen with and without S-9 metabolic activation. The cultures with activation were tested at concentrations ranging from 75 μg/ml to 275 μg/ml; cultures without activation were tested at concentrations ranging from 60 μg/ml to 110 μg/ml.

The results indicated that the test material did not induce a significant increase in the mutant frequencies of cultures tested either with or without metabolic activation. Under the conditions tested the test material was not mutagenic in this screen.

In vivo:

Based on the results of the dose selection study, the maximum tolerated dose was estimated as >5000 mg/kg.

The test article did not induce a statistically significant increase in micro-nuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse micronucleus assay.

To further evaluate the genotoxicity potential as well as justify the applied read across from CAS 122384-85-4, OASIS TIMES v.2.27.15.146 was used to predict whether chromosomal aberrations may occur. The suitability of OASIS TIMES was reviewed in a JRC Scientific and Technical Report (Serafimova, Gatnik, and Worth, 2010) where it was found to have advantages over other predictive models for genotoxicity as it incorporates metabolism.

 

To start, UVCB G Graph 1.0 was used to create a Generic SMILES to incorporate all variations possible in the UVCB nature of the registered substance (EC 272-234-3). Over 1500 isomers were predicted and a filter was used to reduce the number to a manageable but representative amount of structures to be predicted by TIMES. The filter option selected was Molecular Weight with 3 intervals in order to separate the isomers based on the amount of sulfur bridging (1-3). Five members were randomly selected from each distribution group for a total of 15 constituents for final modeling. 

 

The difference between the registered substance (high overbased) and the substance used for read across (CAS 122384-85-4; low overbased) is the degree of calcium carbonate overbasing. Based on the principles in “Specific Rules of Ionic Characteristics of Metal Salts of Organic Chemicals” found in the OASIS software, dissociation of the carbonate group could occur based on the large differences in electronegativity resulting from the ionic bond between the calcium and oxygen ions. Based on the principle that dissociation will occur at the Ca-O bond, both the registered substance and read-across substance would be expected to dissociate to the same phenol, thiobis substance. Therefore, TIMES was modeled with and without hydrolysis selected to mimic dissociation and non-dissociation structures, respectively.

 

Running the model without dissociation (calcium carbonate present) resulted in negative predictions for the parent structures. However, the corresponding parent structures with the calcium dissociated yielded metabolites that were positive in situ for chromosomal aberration due to aromatic ring hydroxylation and the potential formation of hydroquinones and catechols and subsequent oxidation to benzoquinones. However, all of the predicted positive metabolites are below 0.05 for probability to obtain and probability to be. Therefore, the metabolites would not be predicted to be available to interact with DNA. This is consistent with the negative micronucleus study for CAS 122384-85-4 and transformation to genotoxic species does not appear to be relevant in an animal model. 

 

As stated, TIMES was run with hydrolysis turned off to show the prediction with the carbonate group attached. This resulted in negative predictions for both parents and metabolites. This illustrates that dissociation of the calcium carbonate is required for transformation to genotoxic metabolites in situ. This is likely due to the calcium carbonate preventing transformation to a quinone species. Therefore, read across from low overbased to high overbased substances could be considered conservative

Justification for classification or non-classification

The results for the key parameters chosen for genetic toxicity were negative and so the criteria set out in Directive 67/548/EEC and also Regulation (EC) no 1272/2008 do not apply, therefore classification for genetic toxicity was not considered to be necessary.