Terphenyl, hydrogenated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Terphenyl, hydrogenated was studied in vitro for bacterial reverse mutation, mammalian forward mutation and unscheduled DNA synthesis, and in vivo for chromosomal aberration. All studies were negative, demonstrating that there were no indications of genotoxicity.

Link to relevant study records

Reference

Reference 1

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliance study report, available as unpublished report, no restrictions, adequate for assessment

Principles of method if other than guideline:

Method: other: Pharmakon Research International, Inc. method

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphorybosil transferase (HGPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were plated in plastic tissue culture flasks in 5 mL of medium F12FCM5
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction

Test concentrations with justification for top dose:

25, 50, 75, 100 and 300 µg/ml

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

With and without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Remarks:

With and without metabolic activation

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 16-24 hours
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (reduction in colony forming ability of the cells)

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Cytotoxicity was not observed up to 1000 µg/ml.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation: a precipitation of the test article was apparent in the treatment media at all doses above 100 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
Doses tested: 10, 50 and 100 µg/mL with and without metabolic activation.
There were no statistically significant increases in the mutation frequencies of the Terphenyl, hydrogenated treated cultures when compared to the negative controls at the doses and S-9 concentrations tested.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was not observed up to 1000 µg/mL.

Remarks on result:

other: all strains/cell types tested

Conclusions:

It is concluded that Terphenyl, hydrogenated failed to induce mutations in Chinese hamster Ovary (CHO) cells at 25, 50, 75, 100 and 300 μg/mL with and without metabolic activation.

Executive summary:

A key study was performed with Terphenyl, hydrogenated for the cell mutation assay in Chinese hamster Ovary (CHO) cells at 25, 50, 75, 100 and 300 μg/mL with and without metabolic activation. The study was conducted on the hypoxanthine-guanine phosphorybosil transferase (HGPRT) target gene. Cytotoxicity was not observed up to 1000 μg/mL, however precipitation of the test article was apparent in the treatment media at all doses above 100 μg/mL. There were no statistically significant increases in the mutation frequencies of the Terphenyl, hydrogenated treated cultures when compared to the negative controls at the doses and S-9 concentrations tested.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliance study report, available as unpublished report, no restrictions

Principles of method if other than guideline:

Method: other: SRI International method

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

hepatocytes: from rats

Metabolic activation:

with and without

Test concentrations with justification for top dose:

0.1, 0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 µg/mL

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

IUCLID4 Type: Unscheduled DNA synthesis

Species / strain:

hepatocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not observed up to 5000 µg/mL

Remarks on result:

other: all strains/cell types tested, primary rat hepatocytes

Conclusions:

It is concluded that Terphenyl, hydrogenated failed to induce DNA damage and/or repair in an in vitro UDS study with primary rat hepatocyte cultures at concentrations up to 1000 μg/mL.

Executive summary:

A key study for DNA damage and/or repair was performed with Terphenyl, hydrogenated in an in vitro UDS study with primary rat hepatocyte cultures at concentrations of 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 μg/mL with and without metabolic activation. Cytotoxicity was not observed up to 5000 μg/mL. The study was negative.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, predates GLP, minor restrictions in reporting, but otherwise adequate for assessment.

Qualifier:

according to guideline

Guideline:

other: Ames Method (Mutation Research, 1975, vol. 31, pp.347-364)

Deviations:

yes

Remarks:

For sample Terphenyl, hydrogenated in the TA1537 with and without microsomal activation at 10 µL per plate there is an additional data point for the statistical calculations. This additional number does not significantly alter the results.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

0.01, 0.04, 0.2, 1.0, 3.0, 10.0 µL/plate

Vehicle / solvent:

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

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

other: sodium nitrite

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 3

Statistics:

Statistical analysis for significance of difference between treatments and controls is performed for each treatment using a t-test. Values of revertants/plate are transformed using log to the base 10. Variance is calculated as within-levels pooled variance for the treatment and solvent control revertants/plate. Values of p are calculated for a one-sided t-test.

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

Remarks:

Salmonella tester strain TA100 was used in the absence and the presence of metabolic activation, a maximum concentration of 100 µl per plate.

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A toxic screen was run with the test compound at concentrations of 100.0, 30.0, 10.0, 1.0, 0.3 and 0.1 µL per plate using Salmonella tester strain TA100. In the absence and the presence of metabolic activation, a maximum concentration of 100 µL per plate was tolerated by the bacteria without toxic effects.

Remarks on result:

other: all strains/cell types tested, Salmonella typhimurium strains TA1535, TA1538, TA100, TA98

Conclusions:

It is concluded that Terphenyl, hydrogenated failed to induce mutations in 4 strains of Salmonella thyphimurium, when tested up to concentrations close to the toxic range, in the absence and presence of a rat liver metabolic activation system. The test substance is hence considered to be negative for mutagenicity

Executive summary:

A study for bacterial mutagenicity was performed with Terphenyl, hydrogenated in an Ames reverse mutation test with 4 Salmonella thyphimurium strains (TA 1535, TA 1537, TA 98 and TA 100) at test concentration of 0.01, 0.04, 0.2, 1.0, 3.0, 10.0 μL/plate with and without metabolic activation. A toxic screen was run with the test compound at concentrations of 100.0, 30.0, 10.0, 1.0, 0.3 and 0.1 μL per plate using Salmonella tester strain TA100. In the absence and the presence of metabolic activation, a maximum concentration of 100 μL per plate was tolerated by the bacteria without toxic effects. All strains were tested to be negative for mutagenicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Terphenyl, hydrogenated was studied in vitro for bacterial reverse mutation, mammalian forward mutation and unscheduled DNA synthesis, and in vivo for chromosomal aberration. All studies were negative, demonstrating that there were no indications of genotoxicity.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliance study report, available as unpublished report, no restrictions, adequate for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Principles of method if other than guideline:

Method: other: SRI International method

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories Inc., 1180C Day Road, Gilroy, CA 95020
- Weight at study initiation: 175-275 g
- Assigned to test groups randomly: yes
- Housing: two or three per cage
- Diet (e.g. ad libitum): ad libitum (Certified Purina Lab Chow (#5002C))
- Water (e.g. ad libitum): ad libitum (Purified water)
- Acclimation period: minimum 5 days

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Lot/batch no. (if required): 790670

Duration of treatment / exposure:

Single administration

Frequency of treatment:

Not applicable

Post exposure period:

Immediately after test article administration, near the end of the working day and (when appropriate) each day thereafter prior to sacrifice.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Dose / conc.:

2 500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

number of animals: 18 animals per sex per dose
number of animals per sampling time: 6 animals per sex per dose

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

triethylenemelamine (TEM, CAS n° 51-18-3)
- Route of administration: intraperitoneal injection
- Doses / concentrations: 0.2 mg/kg TEM dissolved in Hanks' balanced salt solution.
- Lot/batch no.: 9558

Tissues and cell types examined:

Target cells: bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the dose range-finding and the pilot studies

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
sampling times: 6, 12, 24 hours after dosing

DETAILS OF SLIDE PREPARATION:
Slides were prepared by dropping cell suspensions onto water-dipped slides, passing the slides through the flame of an alcohol lamp several times, and checking the cell density using a phase-contrast microscope. If cell density was too thick, more fixation was added to dilute the suspension. Based on the amount of cell suspension, six or fewer slides were prepared from each tube.
The slides were placed in 7% Giemsa (Gurr's R66 in m/15 Sorensen's buffer, pH 6.8) for 5 to 20 min. rinsed in distilled water, soaked in xylene, and mounted with Permount to make permanent slides for scoring under bright-field or phase-contrast optics (100x oil objective).

Evaluation criteria:

A test article was considered to have elicited a positive response in the in vivo bone marrow cytogenetic assay if either the mean aberrant cell frequency or the mean chromosomal aberration frequency per cell, or both, was significantly greater (P<0.05) in the test article-treated animals than in the negative control animals.

Statistics:

The following statistics were calculated for each animal: MI, the total number of chromosomal aberrations and the frequency of chromosomal aberrations per cell, the number and frequency of aberrations is each category, the number and frequency of cells with structurally aberrant chromosomes, the number and frequency of aneuploid (hyperploid) cells, the number and frequency of polyploid cells, and the number and frequency of several damaged cells.
These statistics have been analysed by the Bartlett's method, a one-way analysis of variance, the Dunnett's test and the Student's t-test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 8, 40, 200, 1000 and 5000 mg/kg
- Clinical signs of toxicity in test animals:
At doses of 8, 40, 200 and 1000 mg/kg: There was no significant effect on body weight, no significant clinical signs of toxicity and no animals died.
At 5000 mg/kg: One male exhibited rough fur, humped backs, red exudate from the nose, slight diarrhea until it died. Another male exhibited rough fur and humped backs until the 14 days of the study.
At 5000 mg/kg: Females exhibited rough fur, humped backs, marked diarrhea and red exudate from the nose and the eyes. One female died.

RESULTS OF 24-HOUR PILOT STUDY
- Dose range: 0, 312, 625, 1250, 2500 and 5000 mg/kg
- Mitotic indices: no significant effect
- Clinical signs of toxicity in test animals:
At 2500 and 5000 mg/kg: Males and females exhibited negative changes in body weight, rough fur and humped backs.
No significant signs of toxicity were observed for animals treated with lower doses.

RESULTS OF 14-DAY PILOT STUDY
- Dose range: 312, 625, 1250, 2500 and 5000 mg/kg
- Clinical signs of toxicity in test animals:
At 5000 mg/kg: 4/5 males and 4/5 females died during the study. clinical signs of toxicity, rough fur, humped back, diarrhea, red exudates from the eyes and nose, slight hyperpnea
At 2500 mg/kg: weight gain reduction, clinical signs of toxicity, rough fur

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals:
At 250 mg/kg bw: no effects.
At 1250 mg/kg bw: a slight exudat from the left eye observed in one female.
At 2500 mg/kg bw: no effects; NOAEL.

Cytogenetic evaluation of bone marrow cells from female rats treated with Terphenyl, hydrogenated: 24-hour sacrifice

	Negative control	2500 mg/kg Terphenyl, hydrogenated	0.2 mg/kg
Number of animals	5	4	5
Mitotic Index (Mean % ±)	5.05 ± 0.44	6.36 ± 1.07	3.31 ± 0.34
Number of cells analyzed	300	240	300
Number of aberrant cells (Mean % ±)	0	2(0.83±0.83)	85(28.33±5.75)
Number of cells with structurally abnormal chromosomes (Mean % ±)	0	2(0.83±0.83)	82(27.33±5.52)
Number of cells (%) with:
Chromosome deletions	0	0	1 (0.40)
Chromosome exchanges	0	0	0
Chromatid deletions	0	2(0.83)	27 (10.89)
Chromatid exchanges	0	0	12 (4.84)
Aneuploidy	0	0	2 (0.81)
Polyploidy	0	0	1 (0.40)
Severe damage	0	0	52 (17.33)
Types of aberrations per cells:
Overall frequency of aberrations (Mean % ±)	0	3 (0.012)	329(1.097±0.267)
Chromosome deletions	0	0	1 (0.004)
Chromosome exchanges	0	0	0
Chromatid deletions	0	3 (0.012)	51 (0.206)
Chromatid exchanges	0	0	14 (0.056)
Number of cells (%) with:
Chromatid gaps	0	1 (0.42)	4 (1.61)
Chromosome gaps	0	0	0

 

Cytogenetic evaluation of bone marrow cells from male rats treated with Terphenyl, hydrogenated: 24-hour sacrifice

	Negative control	2500 mg/kg Terphenyl, hydrogenated	0.2 mg/kg
Number of animals	5	5	4
Mitotic Index (Mean % ±)	5.82 ± 0.31	7.22 ± 0.98	4.79 ± 0.65
Number of cells analyzed	300	300	300
Number of aberrant cells (Mean % ±)	1(0.33±0.33)	1(0.33±0.33)	73(24.33±3.71)
Number of cells with structurally abnormal chromosomes (Mean % ±)	0	0	72(24.0±3.6)
Number of cells (%) with:
Chromosome deletions	0	0	0
Chromosome exchanges	0	0	1 (0.38)
Chromatid deletions	0	0	32 (12.08)
Chromatid exchanges	0	0	14 (5.28)
Aneuploidy	1 (0.33)	0	1 (0.38)
Polyploidy	0	1 (0.33)	0
Severe damage	0	0	35 (11.67)
Types of aberrations per cells:
Overall frequency of aberrations (Mean % ±)	1(0.003±0.003)	1(0.003±0.003)	257(0.857±0.112)
Chromosome deletions	0	0	0
Chromosome exchanges	0	0	1 (0.004)
Chromatid deletions	0	0	65 (0.245)
Chromatid exchanges	0	0	15 (0.57)
Number of cells (%) with:
Chromatid gaps	2 (0.67)	0	7 (2.64)
Chromosome gaps	0	0	0

Conclusions:

Interpretation of results: negative
Terphenyl, hydrogenated does not induce chromosomal damage in male or female Fischer-344 rats under the conditions used in this study.

Executive summary:

This study assessed the ability of Terphenyl, hydrogenated administered by intraperitoneal injection to induce chromosomal damage in bone marrow cells of Fischer-344 rats. In the definitive study, rats were given Terphenyl, hydrogenated at doses of 0, 250, 1250, and 2500 mg/kg body weight. Groups of animals were sacrificed 6, 12, and 24 hr after treatment. The positive control groups received triethylenemelamine (0.2 mg/kg body weight 1 by intraperitoneal injection and were sacrificed 24 hr after treatment. Cells from animals exposed to 0 and 2500 mg/kg and those from animals in the positive control groups were evaluated microscopically for mitotic indexand chromosomal abnormalities. On the basis of the results, it was concluded that Terphenyl, hydrogenated does not induce chromosomal damage in male or female Fischer-344 rats under the conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Key studies were available for in vitro studies including bacterial reverse mutation, mammalian forward mutation and unscheduled DNA synthesis, as well for in vivo micronucleus testing.

A key study for bacterial mutagenicity was performed with hydrogenated terphenyl in an Ames reverse mutation test with 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98 and TA 100) at test concentration of 0.01, 0.04, 0.2, 1.0, 3.0, 10.0 μL/plate with and without metabolic activation (Kulik & Ross, 1978). A toxic screen was run with the test compound at concentrations of 100.0, 30.0, 10.0, 1.0, 0.3 and 0.1 μL per plate using Salmonella tester strain TA100. In the absence and the presence of metabolic activation, a maximum concentration of 100 μL per plate was tolerated by the bacteria without toxic effects. All strains were tested to be negative for mutagenicity. The study was conducted according to OECD 471 guidelines, and was considered to be reliable, adequate and relevant.

A key study for the cell mutation assay in Chines Hamster Ovary (CHO) cells was performed with hydrogenated terphenyl at 25, 50, 75, 100 and 300 μg/mL with and without metabolic activation (Godek et al., 1985). The study was conducted equivalent to OECD 476 on the hypoxanthine-guanine phosphorybosil transferase (HGPRT) target gene according to GLP guidelines, and was considered to be reliable, adequate and relevant. Cytotoxicity was not observed up to 1000 μg/mL, however precipitation of the test article was apparent in the treatment media at all doses above 100 μg/mL. There were no statistically significant increases in the mutation frequencies of the hydrogenated terphenyl treated cultures when compared to the negative controls at the doses and S-9 concentrations tested.

A key study for DNA damage and/or repair was performed with hydrogenated terphenyl in an in vitro UDS study with primary rat hepatocyte cultures at 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 μg/mL with and without metabolic activation (Steinmetz and Mirsalis, 1985). The study was conducted according to GLP guidelines, and was considered to be reliable, adequate and relevant. Cytotoxicity was not observed up to 5000 μg/mL. The study was negative.

A key study for in vivo chromosome aberration was performed with hydrogenated terphenyl in the micronucleus assay. The study was conducted according to OECD 475 and GLP guidelines, and was considered to be reliable, adequate and relevant. This study assessed the ability of hydrogenated terphenyl administered by intraperitoneal injection to induce chromosomal damage in bone marrow cells of Fischer-344 rats. In the definitive study, rats were given hydrogenated terphenyl at doses of 0, 250, 1250, and 2500 mg/kg body weight. Groups of animals were sacrificed 6, 12, and 24 hr after treatment. The positive control groups received triethylenemelamine (0.2 mg/kg body weight 1 by intraperitoneal injection and were sacrificed 24 hr after treatment. Cells from animals exposed to 0 and 2500 mg/kg and those from animals in the positive control groups were evaluated microscopically for mitotic index and chromosomal abnormalities. On the basis of the results, it was concluded that hydrogenated terphenyl does not induce chromosomal damage in male or female Fischer-344 rats under the conditions used in this study.

Supporting studies (Klimisch 4) were also available for bacterial reverse mutagenicity and DNA damage and/or repair. A study for bacterial mutagenicity was performed with Terphenyl, hydrogenated in an Ames reverse mutation test with 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98 and TA 100) with and without metabolic activation (Clark et al., 1979). All strains were tested to be negative for mutagenicity up to 10.000 µg/plate with and without metabolic activation. There were no signs of cytotoxicity. A study for DNA damage and/or repair was performed with terphenyl, hydrogenated (NBP2087922) in an in vitro UDS study with primary rat hepatocyte cultures (Monsanto, 1982) .The test was negative.

Justification for classification or non-classification

The endpoint does not meet classification criteria according to EU and/or CLP criteria.