2-Propenoic acid, reaction products with dipentaerythritol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

DPHA was tested in a reverse mutation (Ames) assay and found to be positive only in S. typhimurium strain TA1535 in the presence of metabolic activation. No mutagenicity was observed in the other S. typhimurium strains (TA1537, TA100 or TA98) or the Escherichia coli WP2uvrA with or without metabolic activation. The read-across substance PETIA was positive in an in vitro mammalian cell gene mutation test conducted according to a method equivalent or similar to OECD Guideline 476 (TK locus). This result was however not considered to reflect a mutagenic potential for DPHA. Indeed, a review of over 200 short-term in vitro and in vivo mutagenicity studies for acrylates and metacrylates showed that this class of substances produces a consistent positive response when tested in the mouse lymphoma assay and/or other in vitro mammalian cell assays designed to detect clastogenicity (Johannsen et al., 2008). This appears to be related to effects on chromosomal numbers and structure rather than direct induction of point mutation. Significant cytotoxicity is observed, generally with a non-linear dose-response both with and without activation. When PETIA was tested according to OECD 476 but with the HPRT gene as target, the substance was negative for genotoxicity.
The results of in vitro mouse lymphoma assays, raising the concern of mutagenicity, are clarified by in vivo testing. The behavior of this class of substances (including DPHA) in the mouse micronucleus test and the in vivo chromosomal aberration assay indicates that they are non-genotoxic in whole animals. In conclusion, a wide body of evidence support the conclusion that DPHA and other do not pose in vivo mutagenic risk. This is in line with observations of non-tumorigenic activity from a series of rodent bioassays conducted with acrylic acid and several acrylate esters (Johannsen et al., 2008).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 April, 2012 to 4 May, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 10, 33, 100, 333 and 1000 µg/plate
Experiment 2:
Without and with S9-mix: 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test substance was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 : 650 µg/plate in DMSO for TA100

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9: 10 µg/plate in DMSO for WP2uvrA

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9: 5 µg/plate in saline for TA1535

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA1537, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at dose levels of 1000 µg/plate and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Dose range finding test:

Precipitate: Precipitation of DPHA on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 μg/plate and upwards.

Toxicity: To determine the toxicity of DPHA, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutagenicity: In the dose range finding test, no increase in the number of revertants was observed upon treatment with DPHA under all conditions tested.

Conclusions:

Under the study conditions, it was concluded that the test substance at concentrations up to 1000 μg/plate is mutagenic in tester strain TA1535 with metabolic activation only. No mutagenicity was observed with or without metabolic activation using other Salmonella typhimurium tester strains (TA1537, TA100 or TA98) and Escherichia coli strain.

Executive summary:

A study was conducted to determine the mutagenic potential of test substance DPHA in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat) according to the OECD Guideline 471 and EU Method B.13/14 in compliance with GLP. DPHA was tested with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix. In the dose range finding test, the test substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance precipitated on the plates at dose levels of 1000 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 10 to 1000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance precipitated on the plates at the top dose of 1000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the presence of S9-mix, the test substance induced 3.6 and 5.8-fold increases in tester strain TA1535 in the first and second experiment, respectively. The increases observed were above the laboratory historical control data range and the increases were greater than three times the concurrent control, were dose-related and observed in two independently repeated experiments. Therefore these increases are considered to be biologically relevant and DPHA is considered to be mutagenic in this strain with metabolic activation. All other bacterial strains showed negative responses over the entire dose range (no significant dose-related increase in the number of revertants in two independently repeated experiments). In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, it was concluded that the test substance at concentrations up to 1000 μg/plate is mutagenic in tester strain TA1535 with metabolic activation only. No mutagenicity was observed with or without metabolic activation using other Salmonella typhimurium tester strains (TA1537, TA100 or TA98) and Escherichia coli strain (Verspeek-Rip, 2013).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From August 19, 2014 to October 03, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, 10% FBS (except during 4 hour treatment), neomycin (5 μg/mL) and amphotericin B (1%). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1:
4 h (without S9 mix): 0.0078, 0.016, 0.031*, 0.063*, 0.13*, 0.25*, 0.5*, 0.75 μg/mL
4 h (with S9 mix): 0.94, 1.9, 3.8*, 7.5*, 15.0*, 22.5*, 30.0* μg/mL

Experiment 2:
24 h (without S9 mix): 0.016, 0.031, 0.063*, 0.13*, 0.25*, 0.5*, 0.75*, 1.0 μg/mL
4 h (with S9 mix): 4.0, 8.0, 16.0*, 20.0*, 24.0*, 28.0*, 32.0* μg/mL

* these concentrations were chosen for the mutation rate analysis

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

2-3 days after sub-cultivation stock cultures were trypsinized at 37°C for 5 minutes. Approximately 1.5×106 (single culture) and 5×102 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 h prior to treatment. After 24 h, the medium was replaced with serum-free medium containing the test substance, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. In the second experiment, the cells were exposed to the test substance for 24 h in complete medium, supplemented with 10% FBS, in the absence of metabolic activation.

DETERMINATION OF CYTOTOXICITY
- toxicity was indicated by a reduction of the cloning efficiency.

Evaluation criteria:

A test substance was classified as positive if it induced either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test substance producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points was considered non-mutagenic in this system.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A trend was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological and statistical significance were considered together.

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentrations of the test substance. No precipitation or phase separation occurred up to the maximum concentration with and without metabolic activation following 4 and 24 h treatment.

RANGE-FINDING/SCREENING STUDIES:
The dose range of the first experiment was set according to the data generated in the pre-experiments. The dose range of the second experiment was adjustedaccording to cytotoxicity data generated in the pre-experiments (without metabolic activation) and in the first experiment with metabolic activation. The highest concentration used in the first pre-experiment was 4,240 μg/mL limited by the solubility of the test substance in DMSO and aqueous medium. Test substanceconcentrations between 33.1 μg/mL and 4,240 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation (4 and 24 h treatment). The pre-experiment was not analysable due to exceedingly severe cytotoxicity down to the lowest concentration. Therefore, a second pre-experiment was performed using concentrations from 0.5 to 60 μg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency 1 or cell density below 50% occurred in experiment 1 at 0.50 μg/mL without metabolic activation and at 22.5 μg/mL and above with metabolic activation. In experiment 2, cytotoxic effects were observed at 0.25 μg/mL and above without metabolic activation and at 20.0 μg/mL and above with metabolic activation.

The relative cloning efficiency in experiment I was within a range from 0 to 10% at the maximum analysable concentration in all experimental parts the data was judged as valid as the relative cell density at the first sub-cultivation following treatment remained at or above 10%. No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. The mutant 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 mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.0 up to 20.7 mutants per 10⁶ cells; the range of the groups treated with the test item was from 7.7 up to 34.5 mutants per 10⁶ cells. EMS (150 μg/mL) and DMBA (2.2 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

Based on the read across study, the test substance is considered to be non-mutagenic at the HPRT locus in V79 cells.

Executive summary:

A study was conducted to assess the mutagenic potential of the read across substance PETIA at the HPRT locus in V79 cells of the Chinese hamster, according to OECD Guidelin 476 and EU Method B.17, in compliance with GLP. The assay was performed in two independent experiments. The cells were exposed to the read across substance for 4 h in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 h with and 24 h without metabolic activation. The maximum concentration of the pre-experiment (4,240 μg/mL) was based on the solubility properties of the read across substance in DMSO and aqueous medium. The concentration range of the main experiments was limited by cytotoxic effects. DMSO was used as solvent. Test concentrations were as follows: (a) Experiment 1: 4 h (without S9 mix): 0.0078, 0.016, 0.031*, 0.063*, 0.13*, 0.25*, 0.5*, 0.75 μg/mL ; 4 h (with S9 mix): 0.94, 1.9, 3.8*, 7.5*, 15.0*, 22.5*, 30.0* μg/mL (b) Experiment 2: 24 h (without S9 mix): 0.016, 0.031, 0.063*, 0.13*, 0.25*, 0.5*, 0.75*, 1.0 μg/mL ; 4 h (with S9 mix): 4.0, 8.0, 16.0*, 20.0*, 24.0*, 28.0*, 32.0* μg/mL (*these concentrations were chosen for the mutation rate analysis). No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system (Wollny, 2014). Based on the read across study, the test substance is considered to be non-mutagenic at the HPRT locus in V79 cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

From Aug 3, 1978 to Oct 29, 1978

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase, TK +/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The mouse lymphoma cell line, L5178Y TK+/-, was derived from the Fischer L5l78Y 1ine of Dr. Donald C1ive. Stocks are maintained in liquid nitrogen and laboratory cultures are periodically checked for the absence of mycoplasma contamination by culturing methods. To reduce the negative control frequency (spontaneous frequency) of TK-/- mutants to as low level as possible, cell cultures are exposed to conditions which select against the TK-/phenotype (exposure to methotrexate) and are then returned to normal growth medium for three or more days before use.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Without metabolic activation: 0.0156, 0.0313, 0.0625, 0.1250 and 0.2500 nL/mL (first trial) and at 0.0156, 0.0625, 0.250, 0.375 and 0.500 nL/mL in a second trial
With metabolic activation: 0.125, 0.250, 0.500, 1.000 and 2.000 nL/mL and at 0.25, 0.50, 2.50, 5.00 and 25.00 nL/mL in a second trial

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of test substance in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

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:
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2-3 d to allow recovery, growth and expression of the induced TK-/- phenotype. Cell counts are determined daily and appropriate dilutions are made to allow optimal growth rates.

NUMBER OF CELLS EVALUATED: At the end of the expression period, 3 x 106 cells for each selected dose are seeded in soft agar plates with selection medium and resistant (mutant) colonies are counted after 10 d incubation. To determine the actual number of cells capable of forming colonies, a portion of the cell suspension is also cloned in normal medium (nonselective). The ratio of resistant colonies to total viable cell number is the mutant frequency.

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in growth compared to the growth of untreated cells

Evaluation criteria:

A compound is considered mutagenic in this assay if:
-A dose-response relationship is observed over 3 of the 5 dose levels employed.
-The minimum increase at the low level of the dose-response curve is at least 2.5 times greater than the solvent and/or negative control values.
-The solvent and negative control data are within the normal range of the spontaneous background for the TK locus.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

(at the highest dose in the first trial and at the upper two doses in the second trial)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(only at the highest tested dose in the second trial)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance was warmed to 42°C to obtain the liquid form and then dissolved in DMSO at 500 µL/mL for use in the preliminary cytotoxicity test. Dilutions were performed with DMSO such that final 1:100 dilutions into growth medium yielded an applied concentration range of 5 µL/mL to 0.078 nL/mL. Toxicity was observed at 0.313 nL/mL for non-activation conditions and concentrations of 0.625 nL/mL and higher were completely lethal to the cells within 24 h of treatment. Therefore, the mutation assay was initiated with an applied concentration range of 2 to 0.0039 nL/mL. Excessive toxicity was obtained at 0.5 nL/mL and higher concentrations in the nonactivation portion of the assay, but the test substance was less toxic under activation conditions and 2 nL/mL was not sufficiently toxic to adequately test for any mutagenic activity. Two additional mutation assays were performed - one to extend the activation conditions to higher concentrations of the test substance and the other to confirm the results of the non-nactivation assay in the first trial. Under activation conditions, the highest applied concentration that resulted in sufficient survival of the cells to complete the mutation assay was 25 nL/mL; at 50 nL/mL and above, all the cells were destroyed within 24 h of treatment. The five dose levels chosen for completion of each mutation assay were selected to span the observed range of toxicity to growth with emphasis on the most toxic doses. After the cells were seeded for mutant selection and measurement of viability, the percent relative growth in the treated cultures was found to range from 82.3% to 16. 1% without activation and from 155.8% to 13.7% with activation, all trials inclusive.

Detailed results:

Without activation, the test substance did not induce a significant increase in mutant frequency for four of the five tested doses in the first trial. The minimum increase considered necessary to demonstrate mutagenesis at a given dose level is 2.5-fold over the average of the solvent and untreated negative control values (background frequency). An increase of approx 3.1-fold over background was observed at the highest tested dose of 0.25 nL/mL. In order to determine the repeatability of this positive response, another nonactivation assay was performed for applied concentrations of 0.50 nL/mL to 0.0156 nL/mL. An elevated mutant frequency (about 3.5-fold over background) was observed for the two highest test concentrations (0.375 nL/mLand 0.50 nL/mL). The toxicity associated with a given applied concentration was variable between the two trials, but the mutagenic activity in both trials was associated with treatments that gave a percent relative growth in the 16.1% to 23.1% range. Therefore, the test substance became mutagenic for highly toxic treatments. With activation, the first assay showed no evidence for mutagenic activity for treatments that did not cause a percent relative growth less than about 75% (1.0 to 2.0 nL/mL dose range). The second trial extended the applied concentration range to 25.0 nL/mL,which reduced the percent relative growth to 13.7%. At this highly toxic dose, the mutant frequency was substantially elevated (about 7.2-fold) over the background frequency. The other applied concentrations of 5.0 nL/mL and less were not demonstrably toxic and caused no increase in mutant frequency. Thus, the test substance became mutagenic only for a highly toxic treatment, as in the nonactivation assay, but higher applied concentrations were necessary to achieve the same toxic action in the presence of the S9 metabolic activation mix.

Conclusions:

Based on the result of read across study, the test substance is considered to be genotoxic in the mouse lymphoma forward mutation assay.

Executive summary:

An in vitro mammalian cell gene mutation test was conducted to determine the genotoxic potential of the read across substance, PETIA according to the method equivalent or similar to the OECD Guideline 476, using mouse lymphoma L5178Y cells. The read across substance was warmed to 42°C to obtain the liquid form and then dissolved in DMSO at 500 µL/mL for use in the preliminary cytotoxicity test. Toxicity was observed at 0.313 nL/mL for non-activation conditions and concentrations of 0.625 nL/mL and higher was completely lethal to the cells within 24 h of treatment. Excessive toxicity was obtained at 0.5 nL/mL and higher concentrations in the non-activation portion of the assay, but the read across substance was less toxic under activation conditions and 2 nL/mL was not sufficiently toxic to adequately test for any mutagenic activity. Two additional mutation assays were performed - one to extend the activation conditions to higher concentrations of the read across substance and the other to confirm the results of the non-activation assay in the first trial. Without activation, the read across substance did not induce a significant increase in mutant frequency for four of the five tested doses in the first trial. The minimum increase considered necessary to demonstrate mutagenesis at a given dose level is 2.5-fold over the average of the solvent and untreated negative control values (background frequency). An increase of approximately 3.1-fold over background was observed at the highest tested dose of 0.25 nL/mL. In order to determine the repeatability of this positive response, another nonactivation assay was performed for applied concentrations of 0.50 nL/mL to 0.0156 nL/mL. An elevated mutant frequency (about 3.5-fold over background) was observed for the two highest test concentrations (0.375 nL/mLand 0.50 nL/mL). The toxicity associated with a given applied concentration was variable between the two trials, but the mutagenic activity in both trials was associated with treatments that gave a percent relative growth in the 16.1% to 23.1% range. Therefore, the test substance became mutagenic for highly toxic treatments. With activation, the first assay showed no evidence for mutagenic activity for treatments that did not cause a percent relative growth less than about 75% (1.0 to 2.0 nL/mL dose range). The second trial extended the applied concentration range to 25.0 nL/mL,which reduced the percent relative growth to 13.7%. At this highly toxic dose, the mutant frequency was substantially elevated (about 7.2-fold) over the background frequency. The other applied concentrations of 5.0 nL/mL and less were not demonstrably toxic and caused no increase in mutant frequency. Thus, the read across substance became mutagenic only for a highly toxic treatment, as in the nonactivation assay, but higher applied concentrations were necessary to achieve the same toxic action in the presence of the S9 metabolic activation mix. Overall, the read across substance induced an increase in mutations at the TK locus in L5178Y mouse lymphoma cells at applied concentrations of 0.25 to 0.5 nL/mL without activation and at 25.0 nL/mL with microsomal activation. The mutagenic activity was associated with highly toxic treatments, and higher applied concentrations were required to achieve the same toxicity in the presence of microsomal activation mix (Brusick, 1979). Based on the results of the read across study, the test substance is considered to be genotoxic in the mouse lymphoma forward mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on the study results, the test substance was considered to be non-clastogenic in the mouse micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 Aug, 2012 to 12 Oct, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: 6 or 7 weeks old
- Weight at study initiation: 31.0 – 35.8 g
- Assigned to test groups randomly: Animals were assigned to these groups using a randomization procedure based on equalization of group mean body weights. Following randomization, animals were identified by sequentially numbered ear tags assigned to each animal during randomization process.
- Housing: Mice were housed up to five per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. The purpose of this system was to supply uninterrupted positive air to each individual rodent Micro-Barrier cage and to capture the effluent air from each cage and re-filter the air (HEPA) prior to introducing the air back into the cage. Heat-treated Sani-Chip hardwood chips were used for bedding to absorb liquids (P.J. Murphy Forest Products, Montville, NJ). Bedding was analyzed by the Manufacturer for any contaminants.
- Diet: Harlan 2018C Certified Global Rodent Diet was provided ad libitum
- Water: Tap water was provided ad libitum
- Acclimation period: 5 or 11 d

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3°F
- Humidity: 50 ± 20%
- Air changes: 10 changes of fresh HEPA-filtered air every hour.
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Concentration of test material in vehicle: 0.1, 1, 10, 100, and 200 mg/mL for dose-range finding study and 50, 100, and 200 mg/mL for definitive micronucleus assay
- Lot/batch no. (if required): MKBD6671
- Purity: Corn oil is characterized as per the Certificate of Analysis on file with the testing facility

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance dose formulations were prepared fresh for each phase of the study prior to use in dose administration. All formulations for the dose-range finding assay (0.1, 1, 10, 100, and 200 mg/mL) and all formulations for the definitive micronucleus assay (50, 100, and 200 mg/mL) were prepared as follows: An appropriate amount of the test substance was combined with an appropriate volume, 80% of the target volume, of the vehicle. Each formulation was vortexed for 2 min and stirred for 10 min using a magnetic stir bar/plate. Remaining volume of the vehicle was added to reach the final targeted volume and each formulation was stirred for an additional 10 min. All formulations appeared as yellow solutions. All formulations were stirred using a magnetic stir bar/plate continuously prior to and during dose administration.

Duration of treatment / exposure:

24 or 48 h

Frequency of treatment:

Single dose

Remarks:

Doses / Concentrations:
1, 10, 100, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
(for dose range-finding assay)

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
(for definitive micronucleus assay)

No. of animals per sex per dose:

Dose range-finding assay: Five male and five female mice were exposed to DPHA at 2000 mg/kg bw and two male mice each were exposed to DPHA at 1, 10, 100 or 1000 mg/kg bw
Definitive micronucleus study: Five male mice/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CP)
- Route of administration: Injection
- Doses / concentrations: 5 mg/mL (10 to 50 mg/kg bw)

Tissues and cell types examined:

Polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

-Bone marrow collection and slide preparation: At the scheduled bone marrow collection time, five mice per treatment were euthanized by CO2 asphyxiation verified by toe pinch reflex. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a labeled centrifuge tube containing approx 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at about 100 x g for about 5 min and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Slides were labeled with harvest date, experiment and animal number using lead pencil. Two slides were prepared from each mouse. The slides were air dried and fixed in methanol. One set of slides was stained with a nucleic acid-specific stain, acridine orange, and was used in microscopic evaluation. Second set was packaged for storage until finalization for the report.

-Scoring for micronuclei (bone marrow evaluation): Bone marrow was evaluated by fluorescent microscopy. To control for bias, bone marrow slides were coded using a random number table by an individual not involved with the scoring process. The staining procedure permits the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange PCEs and ghost-like, dark green NCEs, respectively). Slides initially were scanned using medium magnification to locate suitable areas where the cells were well spread and stained. Next, cells were scored using high power oil immersion as follows. The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring was based upon the micronucleated cell, not the micronucleus; thus occasional cells with more than one micronucleus are counted as one micronucleated PCE (mnPCE), not two (or more) micronuclei. At least 2000 PCEs/animal were scored for the presence of micronuclei (mnPCEs) whenever possible. In addition, at least 1000 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity. PCE proportions <20% of vehicle control.

Evaluation criteria:

Criteria for a valid test: The mnPCE frequency of the vehicle controls must be within the historical vehicle control range, and the positive control must induce a significant increase (p≤0.05) in mnPCE frequency as compared to concurrent vehicle control. Five animals /group are available for analysis.
Evaluation of test results: Once the criteria for a valid assay were met, the results were evaluated as follows:
-The test substance is considered to be positive if it induces a significant increase in mnPCE frequency (p≤0.05) at any dose level or sampling time compared to the concurrent vehicle control.
-The test substance is considered to be negative if no significant increase in mnPCE frequency is observed (p> 0.05) compared to the concurrent vehicle control.
-Other criteria may be used in reaching a conclusion about the study results (e.g., magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.).

Statistics:

The frequency of mnPCEs and the proportion of PCEs to total erythrocytes were determined for each animal and treatment group. Statistical significance (p≤0.05) was determined using the binomial distribution (Kastenbaum-Bowman tables).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Transient piloerection was noted in one of the mice at 500 and 1000 mg/kg bw and in all mice at 2000 mg/kg bw following dose administration.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose range finding assay: No mortality was observed in any of the treatment groups. All mice at 1, 10, 100 mg/kg bw appeared normal during the study period. Transient piloerection was noted following dose administration at 1000 and 2000 mg/kg bw and persisted in one of the male mice at 2000 mg/kg bw on study Day 2. No appreciable reductions to the mean group body weights were observed.
Definitive micronucleus study:
-Clinical signs: No mortality was observed in any of the treatment groups. All mice in the control groups appeared normal. Transient piloerection was noted in one of the mice at 500 and 1000 mg/kg bw and in all mice at 2000 mg/kg bw following dose administration.
-Bone marrow evaluation: No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test substance groups relative to the respective vehicle control groups were observed, suggesting that the test substance did not inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups was observed at 24 or 48 h after dose administration (p > 0.05, Kastenbaum-Bowman Tables).

For data tables, refer to the attached pdf under 'attached background material'.

Conclusions:

Under the study conditions, the test substance was considered to be non-clastogenic in the mouse micronucleus assay.

Executive summary:

An in vivo bone marrow micronucleus study was conducted to determine the clastogenic potential of the test substance DPHA according to OECD Guideline 474, in compliance with GLP. The study was conducted in two phases: a dose range finding (DRF) assay that evaluated the toxicity of the test article and a definitive micronucleus assay that evaluated test article’s potential to increase the incidence of micronucleated polychromatic erythrocyte cells (mnPCEs). The test article was formulated in corn oil. In the definitive micronucleus assay, corn oil was also used as the vehicle control article and cyclophosphamide monohydrate (CP), at a dose of 50 mg/kg, was used as the positive control article. In both phases of the study, test and/or control articles were administered at a dose volume of 10 mL/kg body weight by a single oral gavage. Animals were observed for signs of toxicity during the course of each phase of the study. In absence of toxicity data for DPHA, a DRF assay was performed exposing two male mice/ group at 1, 10, 100, 1000 mg/kg, while five male and five female mice were exposed to 2000 mg/kg, the maximum regulatory guideline recommended dose level. In absence of mortality or severe signs of clinical signs of toxicity, a dose of 2000 mg/kg was tested as high dose in the definitive micronucleus assay. Two lower doses at 500 and 1000 mg/kg were also tested. Since no differences in the clinical signs of toxicity were observed, only male mice were used in the definitive micronucleus assay. In the definitive micronucleus assay, mice were dosed either with the control (vehicle) or positive articles or with DPHA at 500, 1000, or 2000 mg/kg and were euthanized at the scheduled time points and the femoral bone marrow was collected. Bone marrow smears (slides) were prepared and stained with acridine orange stain (a nucleic acid specific stain). Bone marrow cells [polychromatic erythrocytes (PCEs)] were examined microscopically for the presence of micronuclei (micronucleated PCEs; mnPCEs). A statistical analysis of data was performed using the Kastenbaum-Bowman Tables (binomial distribution, p ≤ 0.05). In addition, the proportion of PCEs to total erythrocytes (PCEs/EC ratio) was also determined as indicator of test article’s cytotoxicity. In the DRF assay, no mortality was observed in any of the treatment groups. All mice at 1, 10, and 100 mg/kg appeared normal during the study period. Transient piloerection was noted following dose administration at 1000 and 2000 mg/kg and persisted in one of the male mice at 2000 mg/kg on study day 2. In the definitive micronucleus assay, no mortality was observed in any of the treatment groups. All mice in the control article groups appeared normal. Transient piloerection was noted in one of the mice at 500 and 1000 mg/kg and in all mice at 2000 mg/kg following dose administration. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test article groups relative to the respective vehicle control groups were observed, suggesting that the test article did not inhibit erythropoiesis. No statistically significant increase in the incidence of mnPCEs in test article groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration (p >0.05). CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p≤ 0.05). The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol. Under the study conditions, the test substance was considered to be non-clastogenic in the mouse micronucleus assay (Kulkarni, 2013).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro
A study was conducted to determine the mutagenic potential of test substance DPHA in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat) according to the OECD Guideline 471 and EU Method B.13/14 in compliance with GLP. DPHA was tested with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix. In the dose range finding test, the test substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance precipitated on the plates at dose levels of 1000 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 10 to 1000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance precipitated on the plates at the top dose of 1000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the presence of S9-mix, the test substance induced 3.6 and 5.8-fold increases in tester strain TA1535 in the first and second experiment, respectively. The increases observed were above the laboratory historical control data range and the increases were greater than three times the concurrent control, were dose-related and observed in two independently repeated experiments. Therefore these increases are considered to be biologically relevant and DPHA is considered to be mutagenic in this strain with metabolic activation. All other bacterial strains showed negative responses over the entire dose range (no significant dose-related increase in the number of revertants in two independently repeated experiments). In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, it was concluded that the test substance at concentrations up to 1000 μg/plate is mutagenic in tester strain TA1535 with metabolic activation only. No mutagenicity was observed with or without metabolic activation using other Salmonella typhimurium tester strains (TA1537, TA100 or TA98) and Escherichia coli strain (Verspeek-Rip, 2013).

 

An in vitro mammalian cell gene mutation test was conducted to determine the genotoxic potential of the read across substance, PETIA according to the method equivalent or similar to the OECD Guideline 476, using mouse lymphoma L5178Y cells. The read across substance was warmed to 42°C to obtain the liquid form and then dissolved in DMSO at 500 µL/mL for use in the preliminary cytotoxicity test. Toxicity was observed at 0.313 nL/mL for non-activation conditions and concentrations of 0.625 nL/mL and higher was completely lethal to the cells within 24 h of treatment. Excessive toxicity was obtained at 0.5 nL/mL and higher concentrations in the non-activation portion of the assay, but the read across substance was less toxic under activation conditions and 2 nL/mL was not sufficiently toxic to adequately test for any mutagenic activity. Two additional mutation assays were performed - one to extend the activation conditions to higher concentrations of the read across substance and the other to confirm the results of the non-activation assay in the first trial. Without activation, the read across substance did not induce a significant increase in mutant frequency for four of the five tested doses in the first trial. The minimum increase considered necessary to demonstrate mutagenesis at a given dose level is 2.5-fold over the average of the solvent and untreated negative control values (background frequency). An increase of approximately 3.1-fold over background was observed at the highest tested dose of 0.25 nL/mL. In order to determine the repeatability of this positive response, another nonactivation assay was performed for applied concentrations of 0.50 nL/mL to 0.0156 nL/mL. An elevated mutant frequency (about 3.5-fold over background) was observed for the two highest test concentrations (0.375 nL/mLand 0.50 nL/mL). The toxicity associated with a given applied concentration was variable between the two trials, but the mutagenic activity in both trials was associated with treatments that gave a percent relative growth in the 16.1% to 23.1% range. Therefore, the test substance became mutagenic for highly toxic treatments. With activation, the first assay showed no evidence for mutagenic activity for treatments that did not cause a percent relative growth less than about 75% (1.0 to 2.0 nL/mL dose range). The second trial extended the applied concentration range to 25.0 nL/mL,which reduced the percent relative growth to 13.7%. At this highly toxic dose, the mutant frequency was substantially elevated (about 7.2-fold) over the background frequency. The other applied concentrations of 5.0 nL/mL and less were not demonstrably toxic and caused no increase in mutant frequency. Thus, the read across substance became mutagenic only for a highly toxic treatment, as in the nonactivation assay, but higher applied concentrations were necessary to achieve the same toxic action in the presence of the S9 metabolic activation mix. Overall, the read across substance induced an increase in mutations at the TK locus in L5178Y mouse lymphoma cells at applied concentrations of 0.25 to 0.5 nL/mL without activation and at 25.0 nL/mL with microsomal activation. The mutagenic activity was associated with highly toxic treatments, and higher applied concentrations were required to achieve the same toxicity in the presence of microsomal activation mix (Brusick, 1979). Based on the results of the read across study, the test substance is considered to be genotoxic in the mouse lymphoma forward mutation assay.

 

A study was conducted to assess the mutagenic potential of the read across substance PETIA at the HPRT locus in V79 cells of the Chinese hamster, according to OECD Guideline 476 and EU Method B.17, in compliance with GLP. The assay was performed in two independent experiments. The cells were exposed to the read across substance for 4 h in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 h with and 24 h without metabolic activation. The maximum concentration of the pre-experiment (4,240 μg/mL) was based on the solubility properties of the read across substance in DMSO and aqueous medium. The concentration range of the main experiments was limited by cytotoxic effects. DMSO was used as solvent. Test concentrations were as follows: (a) Experiment 1: 4 h (without S9 mix): 0.0078, 0.016, 0.031*, 0.063*, 0.13*, 0.25*, 0.5*, 0.75 μg/mL ; 4 h (with S9 mix): 0.94, 1.9, 3.8*, 7.5*, 15.0*, 22.5*, 30.0* μg/mL (b) Experiment 2: 24 h (without S9 mix): 0.016, 0.031, 0.063*, 0.13*, 0.25*, 0.5*, 0.75*, 1.0 μg/mL ; 4 h (with S9 mix): 4.0, 8.0, 16.0*, 20.0*, 24.0*, 28.0*, 32.0* μg/mL (*these concentrations were chosen for the mutation rate analysis). No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system (Wollny, 2014). Based on the read across study, the test substance is considered to be non-mutagenic at the HPRT locus in V79 cells.

An in vitro cytogenetic study (according to OECD Guideline 473 or 487) was not conducted since in vivo testing is available (mouse micronucleus study; OECD Guideline 474).

In vivo
An in vivo bone marrow micronucleus study was conducted to determine the clastogenic potential of the test substance DPHA according to OECD Guideline 474, in compliance with GLP. The study was conducted in two phases: a dose range finding (DRF) assay that evaluated the toxicity of the test article and a definitive micronucleus assay that evaluated test article’s potential to increase the incidence of micronucleated polychromatic erythrocyte cells (mnPCEs). The test article was formulated in corn oil. In the definitive micronucleus assay, corn oil was also used as the vehicle control article and cyclophosphamide monohydrate (CP), at a dose of 50 mg/kg, was used as the positive control article. In both phases of the study, test and/or control articles were administered at a dose volume of 10 mL/kg body weight by a single oral gavage. Animals were observed for signs of toxicity during the course of each phase of the study. In absence of toxicity data for DPHA, a DRF assay was performed exposing two male mice/ group at 1, 10, 100, 1000 mg/kg, while five male and five female mice were exposed to 2000 mg/kg, the maximum regulatory guideline recommended dose level. In absence of mortality or severe signs of clinical signs of toxicity, a dose of 2000 mg/kg was tested as high dose in the definitive micronucleus assay. Two lower doses at 500 and 1000 mg/kg were also tested. Since no differences in the clinical signs of toxicity were observed, only male mice were used in the definitive micronucleus assay. In the definitive micronucleus assay, mice were dosed either with the control (vehicle) or positive articles or with DPHA at 500, 1000, or 2000 mg/kg and were euthanized at the scheduled time points and the femoral bone marrow was collected. Bone marrow smears (slides) were prepared and stained with acridine orange stain (a nucleic acid specific stain). Bone marrow cells [polychromatic erythrocytes (PCEs)] were examined microscopically for the presence of micronuclei (micronucleated PCEs; mnPCEs). A statistical analysis of data was performed using the Kastenbaum-Bowman Tables (binomial distribution, p ≤ 0.05). In addition, the proportion of PCEs to total erythrocytes (PCEs/EC ratio) was also determined as indicator of test article’s cytotoxicity. In the DRF assay, no mortality was observed in any of the treatment groups. All mice at 1, 10, and 100 mg/kg appeared normal during the study period. Transient piloerection was noted following dose administration at 1000 and 2000 mg/kg and persisted in one of the male mice at 2000 mg/kg on study day 2. In the definitive micronucleus assay, no mortality was observed in any of the treatment groups. All mice in the control article groups appeared normal. Transient piloerection was noted in one of the mice at 500 and 1000 mg/kg and in all mice at 2000 mg/kg following dose administration. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test article groups relative to the respective vehicle control groups were observed, suggesting that the test article did not inhibit erythropoiesis. No statistically significant increase in the incidence of mnPCEs in test article groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration (p >0.05). CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p≤ 0.05). The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol. Under the study conditions, the test substance was considered to be non-clastogenic in the mouse micronucleus assay (Kulkarni, 2013).

Justification for classification or non-classification

Based on the results of in vitro and in vivo testing, DPHA is not considered to be genotoxic and therefore does not warrant classification according to CLP (EC 1272/2008) criteria.