Phenol, paraalkylation products with C10-15 branched olefins ( C12 rich) derived from propene oligomerization, calcium salts, sulfurized, including distillates (petroleum), hydrotreated, solvent-refined, solvent-dewaxed, or catalyc dewaxed, light or heavy paraffinic C15-C50

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: the registered substance was negative in a GLP guideline (Klimisch code 1) study.

 

Mouse lymphoma- In this key study for in vitro genetic toxicity with an analog (EC 701 -251 -5) (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.

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.

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:

key study

Study period:

18th September to 1st December 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study following GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

UV sensitivity of cultures not verified in all experiments; this deviation from the guideline shown (by historical repeatability of results using the same frozen stock cultures) not to affect the validity of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

no data

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

100, 250, 500, 1000, 5000 and 10000 µg/plate, with and without S-9

Vehicle / solvent:

25% w/w Pluronic F127 (surfactant, CAS # 9003-11-6) in ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: Vehicle was tested as negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See table below.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Bottom agar (25 ml per 15 x 100 mm petri dish) was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956), supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose.

DURATION
- Preincubation period:
Overnight cultures for use in all testing procedures, will be inoculated by transferring a colony from the appropriate master plate to a flask containing culture medium. Inoculated flasks will be placed in a shaker/incubator which will be programmed to begin operation (shaking, 125 ± 25 rpm; incubation, 37 ± 2°C) so that the overnight cultures are in log phase or late log phase when turbidity monitoring begins.
- Exposure duration:
In the plate incorporation methodology, the test article, the tester strain and the S9 mix (where appropriate) were combined in molten agar which was overlaid onto a minimal agar plate. Following incubation at 37 ± 2 °C for 48 ± 8 hr, revertant colonies were counted. All doses of test article, vehicle controls, and positive controls were plated in triplicate.


DETERMINATION OF CYTOTOXICITY
- Method: The growth inhibitory effect (cytotoxicity) of the test article to the test system will be determined in order to allow the selection of appropriate doses to be tested in the mutagenicity assay.

Evaluation criteria:

The following criteria will be used to determine a valid assay:
1.Tester Strain Integrity: Salmonella typhimurium
a. uvrB Excision Repair Deletion: To demonstrate the presence of the ma B excision repair deletion, tester strain cultures must exhibit sensitivity to UV light.
b. rfa Wall Mutation: To demonstrate the presence of the rfa wall mutation, tester strain cultures must exhibit sensitivity to crystal violet.
c. pKM 101 Plasmid: To demonstrate the presence of the R-factor plasmid, pKM101, cultures of tester strains TA98 and TA100 must exhibit resistance to ampicillin.
d. Characteristic Number of Spontaneous Revertants: To demonstrate the requirement for histidine, the tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions.

2. Tester Strain Integrity: Escherichia coli
a. uvrA Excision Repair Deletion: To demonstrate the presence of the uvrA excision repair deletion, tester strains must exhibit sensitivity to UV light.
b. Characteristic Number of Spontaneous Revertants: To demonstrate the requirement for tryptophan, the tester strain culture must exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions. The acceptable range for the WP2uvrA mean vehicle control is 5 to 40 revertants per plate.
3. Tester Strain Culture Density: To demonstrate that appropriate numbers of bacteria are plated, the density of tester strain cultures must be greater than or equal to 0.5 x 10ˆ9 bacteria per ml and/or have reached a target level of turbidity demonstrated to produce cultures with a density greater than or equal to 0.5 x 10ˆ9 bacteria per ml.

4. Positive Control Values
a. Positive Control Values in the Absence of S9 Mix
b. Positive Control Values in the Presence of S9 Mix (S9 Mix Integrity)

5. Cytotoxicity
A minimum of three non-toxic dose levels will be required to evaluate assay data

Statistics:

no data

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA

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:

- Precipitation: Test article precipitate was observed on the plates at 5.00 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic concentration:
With metabolic activation: > 10,000 µg/plate
Without metabolic activation > 10,000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
Doses 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 strains TA 100 and WP2uvrA in both the presence and absence of a 10% S9 mix (one plate per dose). Ten doses of test article, from 5,000 to 6.67 µg per plate, were tested. These data were generated in Experiment 17865-Al. No cytotoxicity was observed up to 5,000 µg per plate in the dose rangefinding study with tester strains TA100 and WP2uvrA in either the presence or absence of S9 mix as evidenced by a normal background lawn and no reduction in the number of revertants per plate. Test article precipitate was observed on the plates at 33.3 µg per plate and above with both tester strains in both the presence and absence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

All test validity criteria were satisfied. There was no evidence of cytotoxicity based on condition of the background lawn or revertants/plate data at doses up to 10,000 µg/plate. The greatest mean number of revertants/plate over concurrent controls among all test doses, all strains and for both assays was 100% (TA 1537, next highest 81%) with S-9 and 100% (TA 1535 and 1537, next highest 47%) without S-9. There was no indication of a dose-related decrease or increase of mean revertants/plate.

Conclusions:

Not mutagenic with or without metabolic activation

Executive summary:

The mutagenic potential of the substance was assessed in an "Ames" test conducted under conditions of GLP and in accordance with OECD guideline 471. The results of the Salmonella - Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that, under the conditions of this study, in both an initial and a confirmatory assay, the test article did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor^TM-induced rat liver (S9). No cytotoxicity was observed up to 10,000 µg/plate with the Salmonella tester strains or with WP2uvrA in either the presence or absence of S9 mix. Test article precipitate was observed on the plates at 5.00 µg/plate. The test is therefore considered to have a negative result and the substance is considered non-mutagenic under the conditions of teh test in both the presence and absence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

See category justification attached in Section 13 for more information. Evaluating alkyl phenate sulfides (“phenates”) as a category is appropriate based on similar chemical structures and starting materials, manufacturing processes, physical and chemical properties, functional uses as a lubricating oil additive, and toxicological data. Regarding the ECHA Read-Across Assessment Framework (2017), the alkyl phenate category fit into Scenario 6 (different compounds with the same effect and no variation in the strength of that effect across substances).

Phenates in this category are manufactured in a similar way and from the same staring alkylphenol, tetrapropenyl phenol (“TPP”, EC 310-154-3; AKA phenol, dodecyl-, branched (PDB, PDDP)). The primary difference among the phenates is if they have calcium carbonate basing; the amount of overbasing may also differ. However, based on the trends observed with the robust toxicology data for the category, the amount of calcium carbonate overbasing is not expected to alter the hazards, especially as the core phenate functionality does not change and calcium carbonate has a low hazard potential.

Based on the data and consistent trends observed among category members, phenates have low hazard for human health and the environment. The registered (target) substance, EC 701-249-4) is very similar to EC 701-251-5 except that it lacks the calcium carbonate overbasing. Therefore, EC 701-251-5 can be used as direct read across (and is used as the test material in the target record, where data exists for this source substance); EC 272-388-1 (no calcium hydroxide added) brackets EC 701-249-4 with EC 701-251-5 regarding different levels of calcium carbonate overbasing.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor S-9 metabolic activation

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>100 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results of the preliminary Toxicity Test (not shown) conducted on the test material indicated significant toxicity at 100 µg/ml for the cultures with and without S-9 metabolic activation. Based on the results of the Toxicity Test, the test material was tested in the Mutagenicity Screen at concentrations ranging from 10 µg/ml to 275 µg/ml with metabolic activation and from 30 µg/ml to 110 µg/ml without activation. After the two-day expression period, six cultures with metabolic activation and six cultures without activation were selected for cloning based on the degree of observed toxicity. The cultures were cloned at 75, 100, 150, 200, 250, and 275 µg/ml (with metabolic activation) and 60, 70, 80, 90, 100, and 110 µg/ml (without activation).

None of the cultures treated with test material with or without metabolic activation exhibited mutant frequencies significantly different from the average mutant frequencies of the corresponding negative, solvent controls. Percent total growth ranged from 32% to 94% (with metabolic activation) and 10% to 95% (without activation). The positive control (DMBA) responded satisfactorily.

Under the conditions tested the test material was not mutagenic to L5178Y TK+/- cells in this assay.

First criterion for a valid test (as stated in the report) was not met without S-9, total growth only 32%. Total cell growth at the high dose without S-9 was 10%. Other test validity criteria were satisfied. The highest mutation frequency over control among all test doses was 4% with S-9 and 51% without S-9. There was no indication of a dose-related increase of mutation frequency with S-9. There was a trend without S-9 to higher mutation rates with progressively lower total growth at doses of 80-110 µg/mL. No statistical analysis was performed on the study results, as they were not needed.

Conclusions:

Under the conditions tested the test material was not mutagenic to L5178Y TK+/- cells in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

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.

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:

experimental study

Adequacy of study:

key study

Study period:

11th September to 11th October 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed per recognized guideline following GLP.

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:
The dose levels in the main study were selected based on results from the dose selection study. the dose levels selected were1250, 2500 and 5000 mg/kg.

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:

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 OECD guideline 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The alkyl phenate sulfide category has multiple negative OECD 471 studies. The study with analog substance EC 701-251-5 did not test all the strains per current guidelines. However, an OECD 471 study with EC 701-249-4, did. The difference between the alkyl phenate sulfides is that EC 701 -208 -0 and EC 701-251-5 have calcium carbonate overbasing whereas EC 701-249-4 has calcium dihydroxide. However, this difference does not change the toxicology as explained in the read across and category assessment attached in section 13. This includes that neither calcium hydroxide nor calcium carbonate are genotoxic (Health Council of Netherlands, 2003; EU, 2008; ECHA BPC, 2016).

In addition, a study with EC 272-388-1 was also negative in an OECD 471 study (OECD SIAR, 2009). EC 272-388-1 does not have calcium hydroxide added and provides additional evidence that the alkyl phenate sulfide molecule is not mutagenic.

The registered substance (EC 701 -249 -4) does not have in vitro mammalian genotoxicity data. Therefore, the OECD 476 study with EC 701-251 -5 is used to fill this gap.

References:

1.      Health Council of the Netherlands. Health-based reassessment of administrative occupational exposure limits for calcium carbonate (CAS 471-34-1). 2003.

2.      European Union (EU). Draft Assessment Report for calcium carbonate. Volume 1. August, 2008.

3.      ECHA Biocidal Products Committee (BPC). Opinion on the application for approval of hydrated lime. April 14, 2016. 

Justification for classification or non-classification

Based on multiple negative genotoxicity studies for the alkyl phenate sulfide category, no classification is warranted.