Triisobutyl phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Triisobutyl phosphate is not mutagenic in the Ames test using Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 (GLP-compliant OECD Guideline 471 study) and in the HPRT gene mutation assay (GLP-compliant OECD Guideline 476 study).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, In den Leppsteinwiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material: Triisobutylphosphate
- BASF Test Item No.: 11/0642-1
- Batch Number: B722 v. 26.09.2011
- Purity: 97.8 g/100 g (± 0.1) determined by 1H-NMR-analysis (for details see analytical report No. 11L00444)
- Expiration Date: September 26, 2013
- Physical state, appearance: Liquid, colourless, clear
- Storage conditions: RT

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

See any other information on material and methods.

Vehicle / solvent:

- Solvent: ethanol
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures. The final concentration of ethanol in the culture medium was 0.5 % (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5×10^6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days.

NUMBER OF REPLICATIONS:
- The study was performed in two independent experiments, using identical experimental procedures.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Acceptability of the assay:
The gene mutation assay is considered acceptable if it meets the following criteria: The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data. The positive control substances should produce a significant increase in mutant colony frequencies. The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.

Evaluation of results:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS GENOTOXICITY:
- No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. In experiment I the induction factor exceeded the threshold of 3.0 at 20.8 µg/mL in the first culture without metabolic activation (induction factor of 3.9). This isolated effect however, was biological irrelevant as it was based on the rather low solvent control of just 6.7 colonies per 10^6 cells. The absolute value of the mutation frequency remained well within the historical range of solvent controls. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of the second experiment with metabolic activation. Since all of the values of the mutation frequency remained within the historical range of solvent controls and the threshold described above was not exceeded the trend was judged as biologically irrelevant.

TEST-SPECIFIC CONFOUNDING FACTORS:
- In experiment I phase separation occurred at 166.0 mg/mL and above in the presence of metabolic activation.
- In the pre-test no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item was observed.

RANGE-FINDING/SCREENING STUDIES:
- In the range finding pre-experiment test item concentrations between 20.8 and 2660 µg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant toxic effects were observed at 332.5 µg/mL and above following 4 hours and treatment with and without metabolic activation. After 24 hours treatment relevant toxic effects occurred at 166.3 µg/mL and above. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Phase separation occurred at 1330.0 µg/mL and above after 4 hours and 24 hours treatment without metabolic activation and at 332.5 µg/mL and above with metabolic activation (4 hours treatment).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in the first experiment at 41.5 µg/mL (culture I) without metabolic activation. Exceedingly severe cytotoxic effects impeded analysis at 83.0 µg/mL and above in the first experiment without metabolic activation and at 120.0 µg/mL and above in the second experiment without metabolic activation. In the first experiment with metabolic activation phase separation of the test item limited the analysable concentration range to 249 µg/mL. In the second experiment with metabolic activation phase separation occurred together with exceedingly severe cytotoxic effects limiting the analysable concentration range to 240 µg/mL.

CONCLUSION:
It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Triisobutylphosphate is considered to be non-mutagenic in this HPRT assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material: Etingal
- Analytical purity: 98%
- Lot/batch No.: P.88
- Storage condition of test material: Room temperature

Target gene:

his

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

20 µg - 5000 µg/plate (SPT);
20 µg - 5000 µg/plate (1st PIT);
15 µg - 5000 µg/plate (2nd PIT)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S-9 mix: 2-aminoanthracene. without S-9 mix: N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylendiamine, 9-aminoacridine chloride monohydrate

Details on test system and experimental conditions:

Standard plate test
Test tubes containing 2 mL portions of soft agar which consists of 100 mL agar (0 .6% agar + 0 .6% NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0 .5 mM biotin) are kept in a water bath at 45°C.
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test
0 .1 mL test solution, 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 mL of soft agar is added and after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds. After incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Statistics:

Not specified

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Standard plate test, with and wthout S-9 mix: no increase in the number of his+ revertants is obeserved in all strains.
Preincubation test, with and wthout S-9 mix: no increase in the number of his+ revertants is obeserved in all strains.
Toxicity: A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed in the standard plate test from about 100 µg/plate (TA 1535) or from about 2500 µg/plate (TA 100) onward; in the preincubation test bacteriotoxicity was observed with all strains at dose ≥ 500 µg/ plate .

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material: Triisobutylphosphate (Nr . 112)
- Physical state: colourless liquid
- Analytical purity: ± 98%
- Purity test date: December 17, 1990
- Lot/batch No.: Abl.-Nr . 89-2977 (P.13)
- Expiration date of the lot/batch: June 01, 1990
- Stability under test conditions: Pure: stable for months. In solvent: at least 12 hours in ethanol, and methanol at least 24 hours in H20
- Storage condition of test material: room temperature, light protected

Target gene:

his

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

10, 33.3, 100, 333.3, 1000, 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the test article was dissolved in ethanol. The solvent was chosen because of its solubility properties and its relative nontoxicity for the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: Without metabolic activation: 4-nitro-o-phenylene-diamine. With metabolic activation: 2-aminoanthracene.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: For the pre-incubation method the test solution, the S9 mix or the S9 substitution puffer, and the bacterias were incubated for 30 minutes at 30°C in a shaking water bath. After this period 2 .0 mL of molten 45°C overlay agar was added to each tube. The mixture was poured on mimimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

For each strain and dose level, including the controls, a minimum of three plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL: Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
500 μL: S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test withoutmetabolic activation),
100 μL: Bacteria suspension (cf . test system, pre-culture of the strains),
2000 μL: Overlay agar

Evaluation criteria:

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
A test article is considered as mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxic effects, evidenced by a partial or complete reduction in the number of revertants, occurred with and without metabolic activation in the plate incorporation test and the preincubation test at the higher dose levels in nearly all strains used.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test article showed normal background growth up to 5000.0 μg/plate with and without S9 mix in all strains used.
Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In a GLP-compliant guideline study Triisobutyl phosphate does not exhibit in vivo mammalian genotoxicity in mouse bone marrow cells.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material: MCS 2518
- Physical state: colorless liquid
- Analytical purity: 99.8%
- Lot/batch No.: MIC-5041454
- Storage condition of test material: room temperature

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., Portage, MI.
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: not specified
- Assigned to test groups randomly: yes, by a computer-generated randomization scheme.
- Fasting period before study: not specified
- Housing: two per cage prior to dosing and one per cage after dosing
- Diet (ad libitum): Purina Certified Laboratory Rodent Chow No. 5002 (Trademark of Purina Mills Inc., St. Louis, Mo.)
- Water (ad libitum): supplied by the public water system of St. Louis, MO
- Acclimation period: minimum of 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-21.1
- Humidity (%): 40-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12-hour light cycle

IN-LIFE DATES: not specified

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: 10 mL/kg bw

Details on exposure:

Animals were treated by a single intraperitoneal injection. Animals were sacrificed for micronucleus evaluation (five animals/sex/group) at 24, 48 and 72 hours after dosing.

Duration of treatment / exposure:

Single treatment

Frequency of treatment:

Doses were administered once.

Post exposure period:

up to 72 hours

Dose / conc.:

300 mg/kg bw (total dose)

Dose / conc.:

600 mg/kg bw (total dose)

Dose / conc.:

1 200 mg/kg bw (total dose)

No. of animals per sex per dose:

Test group: 15
Vehicle control: 15
Positive control: 5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide in saline
- Justification for choice of positive control(s): not specified
- Route of administration: intraperitoneal injection
- Doses / concentrations: 10 mL of solution/kg body weight

Tissues and cell types examined:

Bone marrow from both femora of each animal were pooled for slide preparation.
For each animal, two scorers each evaluated:
- 500 total erythrocytes for polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)
- 500 PCEs for micronucleated polychromatic e rythrocytes (MN PCEs).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dose levels for the main study were selected based on toxicity rangefinding study data. The maximum dose selected for testing in the micronucleus experiment was 1200 mg/kg bw (approximately 69% of the combined calculated LD50 of 1730 mg/kg).

DETAILS OF SLIDE PREPARATION:
All animals were sacrificed by cervical dislocation and their femora were removed. Each bone was opened at the end and the bone marrow was flushed with approximately 2 ml of fetal bovine serum in a centrifuge tube. Bone marrow from both femora of each animal were pooled for slide preparation. The suspension was centrifuged to remove the serum. Portions of the remaining cells were placed on a clean glass microscope slide and a smear was prepared. Two slides were initially prepared for each sample and the remaining cell suspension was stored refrigerated to prepare additional slides if needed. Following preparation of the smears the slides were allowed to air dry overnight. The slides were stained using a HemaTek II slide staining machine and a Wright-Giemsa Stain Pak which includes stain, buffer and rinse solutions.

Evaluation criteria:

To determine whether a statistically significant response in MN PCE frequency is treatment related the following criteria are considered:
- whether there are dose and time-dependent effects that are consistent with a treatment-induced response
- the degree of the response in relation to both concurrent and historical negative and positive control data

Statistics:

Micronucleated PCE frequencies observed for each animal were transformed as the square root prior to analysis (Snedecor and Cochran; 1967, MacGregor et al., 1987). PCE/total erythrocyte ratios were not transformed. A Dunnett's test (one sided) was used for comparison of treatment group and positive control values with vehicle control values (Dunnett, 1955). A critical value of p≤0.05 was used for statistical significance.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the rangefinding experiments, the tes substance was found to be toxic to male and female CD-1 mice at 2000 mg/kg bw and greater as indicated by clinical signs of toxicity to the treated males and deaths in the treated males and females. Using the binomial method, the combined LD50 was determined to be 1730 mg/kg. Based on these results, a target dose of 1200 mg/kg (approximately 69% of the combined LD50 value) was selected as a maximum dose that would insure a reasonable probability of observing signs of toxicity but allow survival of the treated animals through the 72 hour time point. Two additional lower doses (300 and 600 mg/kg bw) were also selected for testing.

RESULTS OF DEFINITIVE STUDY
No deaths were observed in the treatment or control groups. All animals in the treatment and control groups appeared normal throughout the experiment and no statistically significant decreases in mean body weights compared to control group values were observed. No statistically significant decreases in mean PCE/erythrocyte ratios were observed in any of the test substance treatment groups.
No statistically significant increases in micronucleated PCE frequency were observed for test substance dosed groups at any of the sacrifice times. The positive control (cyclophosphamide) yielded expected positive responses in micronucleated PCE frequency indicating the adequacy of the experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro Ames Test

Triisobutyl phosphate was tested in a reverse mutation assay using Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA98 at 15 to 5000 µg/plate (plate incorporation test) according to OECD 471 (BASF, 1990).

The substance was tested in the Ames test (standard plate test and preincubation test) both in the presence and in the absence of a metabolizing system obtained from rat liver (S-9 mix).

An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

A bacteriotoxic effect (reduced his background growth, decrease in the number of his+ revertants) was observed in the standard plate test from about 100 µg/plate (TA 1535) or from about 2500 µg/plate (TA 100) onward; in the preincubation test bacteriotoxicity was observed with all strains at dose ≥ 500 µg/ plate .

According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here.

 

In a second study, the potential of Triisobutyl phosphate to induce gene mutations was tested in the plate incorporation test (exp. I) and the pre-incubation test (exp. II), according to OECD 471 (CCR, 1992). The Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were used in two independent experiments (experiment I = plate incorporation test; experiment II = pre-incubation test), both with and without liver microsomal activation . Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10.0; 33.3; 100.0; 333.3; 1000.0; and 5000.0 μg/plate. Toxic effects, evidenced by a partial or complete reduction in the number of revertants, occurred with and without metabolic activation in experiment I and II at the higher dose levels in nearly all strains used. The plates incubated with the test article showed normal background growth up to 5000.0 μg/plate with and without S9 mix in all strains used. Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frame shifts in the genome of the strains used.

Supportingly, Triisobutyl phosphate was tested in a bacterial reverse mutation assay (Ames test) (Monsanto Company, 1992). Triisobutyl phosphate had no mutagenic activity in this study either with or without metabolic activation. Cytotoxicity occurred in nearly all strains at the higher concentrations both with and without metabolic activation.

In vitro HPRT

In a GLP-compliant gene (HPRT) mutation assay, according to OECD guideline 476, chinese hamster lung fibroblast (V79) were exposed with and without metabolic activation (Harlan, 2012) .The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours.The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum concentration of the pre-experiment (2660 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and phase separation. The test item was dissolved in ethanol. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Triisobutylphosphate is considered to be non-mutagenic in this HPRT assay.

In accordance with column 2 of REACH Annex VIII, in vitro chromosome aberration studies do not need to be conducted as adequate data from in vivo studies are available.

In vivo MNT

The potential for Triisobutyl phosphate to induce chromosomal effects was tested in a mouse bone marrow micronucleus assay, according toEPA OPPTS 870.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay) (Monsanto, 1992). The test substance was administered by intraperitoneal injection to groups of male and female CD-1 mice at target doses of 300, 600 or 1200 mg/kg body weight for the low, mid and high dose groups, respectively. Negative control groups were treated with vehicle only (corn oil, 10 mL/kg bw) and positive control groups were treated with cyclophosphamide (60 mg/kg bw). Mouse bone marrow from test substance treated and vehicle control groups was sampled at 24, 48 and 72 hours after dosing. A single sampling time of 24 hours after dosing was used for the cyclophosphamide positive control group. Slides of bone marrow cells were made from five animals/sex/time point for each group and scored for the occurrence of micronucleated polychromatic erythrocytes and PCE/erythrocyte ratios.

The highest dose level tested, 1200 mg/kg body weight, was 69% of the combined LD50 (1730 mg/kg) estimated from toxicity rangefinder experiments. Triisobutyl phosphate did not induce statistically significant decreases in mean body weights, statistically significant decreases in mean PCE/erythrocyte ratios or increases in the frequency of micronucleated PCEs. Significant increases in mean micronucleated PCE frequencies were observed for the cyclophosphamide treated animals demonstrating the ability of the study conditions to detect micronucleus induction. The observations and findings of this study indicate that Triisobutyl phosphate does not exhibit in vivo mammalian genotoxicity in mouse bone marrow cells under the experimental conditions of this study.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames test, the HPRT test and the in vivo MNT. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No. 2017/776.