Diundecyl phthalate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria. Key study. Experimental study following method equivalent to OECD guideline 471. Diundecyl phthalate (DUP) was not mutagenic with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 with and without metabolic activation.

In vitro gene mutation study in bacteria. Key study. Read across approach. Experimental study following method equivalent to OECD guideline 471. Based on the read-across approach from the analogue substante Ditridecyl phthalate (DTP), DUP was determined to be not mutagenic with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA with and without metabolic activation.

In vitro mammalian chromosome aberration. Key study. Read across approach. Experimental study following method according to recommended guidelines (Dean B., 1984 and Galloway S., 1985) and with international standards (Richardson C., 1989). Based on the read-across approach from the analogue diisononyl phthalate (DINP) assessed in an vitro chromosomal aberration test, DUP is determined to be non-cytogenic to mammalian cells.

In vitro gene mutation study in mammalian cells. Key study: Experimental study following method equivalent to OECD guideline 490. DUP was found negative in the Mouse Lymphoma Assay in the presence and absence of metabolic activation.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

This information was not provided

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from Aroclor 1254-induced male Sprague-Dawley rats (RLI) or male Syrian hamsters (HLI).

Test concentrations with justification for top dose:

Main test (2 experiments): 0 (solvent control), 10, 33, 100, 333, 1000, 3333 and 10000 μg/plate with S9 mix, and 0 (solvent control), 10, 33, 100, 333 and 1000 μg/plate without S9 mix.
The final dose level selection was based on the results of a preliminary range-finding study conducted with TA100 in the presence and absence of S9.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: insoluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamin: -S9: TA98 (5.0 μg/plate); 2-aminoanthracene: +S9: TA100 (1.0 μg/plate), TA1535 (2.5 μg/plate), TA1537 (2.5 μg/plate), and TA98 (1 μg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; Preincubation

DURATION
- Preincubation period: 20 min at 37ºC
- Exposure duration: 48 hours at 37ºC

SELECTION AGENT (mutation assays): the lack of amino-acid (Histidine) in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 2 independent tests with 3 plates/dose/strain.

DETERMINATION OF CYTOTOXICITY
- Method: Not reported but usually by reduced number of spontaneous revertants, microcolony formation, thinning of background lawn.

Evaluation criteria:

A mutagenic response was defined as a reproducible, dose-related increase in the number of histidine-independent colonies over the spontaneous incidence; there was no requirement for a specific magnitude of increase.

Statistics:

No statistical methods were used.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2

Genotoxicity:

other: not tested

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitates were observed at a dose of 10000 μg/plate with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
A preliminary range-finding study was conducted with TA100 in the presence and absence of S9.

Remarks on result:

other: E. coli WP2 not tested

First and second (repeat) experiments yielded similar results, the results presented in the table below are from the second experiment.

Table 1. Results with diundecyl phthalate (lab: SRI; solvent: DMSO)

Dose	TA100						TA1535						TA1537						TA98
	NA		10% HLI		10% RLI		NA		10% HLI		10% RLI		NA		10% HLI		10% RLI		NA		10% HLI		10% RLI
(µg/plate)	mean	SEM	mean	SEM	mean	SEM	mean	SEM	mean	SEM	mean	SEM	mean	SEM	mean	SEM	mean	SEM	mean	SEM	mean	SEM	mean	SEM
0.000	116	7.8	135	6.9	114	6.9	35	4.6	16	1.5	9	2.3	5	1.5	5	0.7	7	2.1	20	3.5	28	2.3	28	0.9
10.000	121	21.2					29	1.8					6	1.5					14	0.3
33.000	96	5.9					31	0.9					7	2.1					17	0.0
100.000	103	6.1	108	12.5	140	9.3	31	2.9	12	2.3	8	0.9	3	0.3	5	1.5	7	0.3	17	2.2	21	4.0	29	2.0
333.000	90	6.7	121	7.2	133	4.8	28	4.8	8	1.2	7	0.7	5	1.5	7	1.7	6	1.5	13	0.7	22	4.7	29	2.6
1000.000	89	13.2	109	2.2	127	12.3	23	4.3	7	0.9	11	2.1	4	1.5	5	0.9	4	1.5	13	0.6	26	3.5	24	3.2
3333.000			99	4.8	114	13.5			9	1.7	6	0.0			5	1.9	6	2.1			26	1.2	23	3.2
10000.000			89 P	9.0	114 P	3.3			12 P	1.7	10 P	1.3			8 P	1.5	6 P	0.6			20 P	3.7	26 P	0.6
POSITIVE	361	3.2	1316	178.5	1046	72.0	449	32.9	564	13.0	209	9.0	181	11.0	357	66.4	210	1.8	876	40.4	1543	78.5	919	31.5

Lab SRI, SRI International

Solvent: DMSO, dimethyl sulfoxide

NA, not activated; 10% HLI, Aroclor 1254-induced Syrian hamster liver S-9; 10% RLI, Aroclor 1254-induced rat liver S-9

p: precipitate present on plates

Conclusions:

Diundecyl phthalate was not mutagenic with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 with and without metabolic activation.

Executive summary:

In a bacterial reverse mutation assay following a method similar to OECD guideline 471, the test item diundecyl phthalate diluted in Dimethylsulfoxide (DMSO) was tested with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 with and without metabolic activation (S9) using the preincubation method. Based on results of a preliminary assay, for the main test two independent experiments and 3 replicates of each were conducted with doses of 0 (solvent control), 10, 33, 100, 333, 1000, 3333 and 10000 μg/plate with S9 mix, and 0 (solvent control), 10, 33, 100, 333 and 1000 μg/plate without S9 mix. Concurrent solvent and positive controls were included in every experiment and their responses were considered valid. Precipitates were observed at a dose of 10000 μg/plate with metabolic activation. The test item did not show mutagenic activity in any of the bacterial strains tested.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

This information was not provided

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):

CELLS USED
- Source of cells: Dr. Donald Clive, Wellcome Research Laboratories.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Medium RPMI 1640
For maintenance of the cell line: RPMI 1640 supplemented with 10% horse serum, pluronic solution, l-glutamine and antibiotics.
Cloning medium: maintenance medium plus 0.35% BBL agar
Selection medium: cloning medium with a final concentration of 3 µg/mL of trifluorothymidine (TFT)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver activation system (S-9)

Test concentrations with justification for top dose:

In the absence of S9: 0.00, 2.00, 4.00, 5.00, 6.00, 8.00 and 10.00 µL/mL
In the presence of S9: 0.00, 1.00, 2.50, 4.00, 5.00, 5.54 and 8.00 µL/mL
Dose levels were selected based on results obtained from a preliminary toxicity test. Dose levels were chosen to produce relative suspension growth values over the entire range from 100% survival to little or no survival.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: DUP poorly soluble in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.

DURATION
- Exposure duration: 4 hours at 37ºC
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 6 x 10^6 cells exposed to the test item, 3 x 10^6 cells to select mutant cells; 200 cells to determine cloning efficiency.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was assessed as ‘percent relative total growth”, obtained by multiplying the relative suspension growth of the cells over the 2-day expression period by the cloning efficiency. Cloning efficiency was defined as growth of the test-material-treated cells in the absence of TFT, relative to non-treated or solvent-control-treated cells in the absence of TFT.

OTHER: colonies were counted at a size setting of 1.3 using an Artek Model 880 counter. The smallest colony counted under these conditions was between 0.2 and 0.3 mm in diameter. Colony size distributions were not performed during this study.

Evaluation criteria:

The minimum criterion necessary to demonstrate a significant positive response in this assay was considered to be a mutant frequency value of 1.5 times the mean negative control value plus 10 X 10-6 together with a dose or toxicity related increase in mutant frequency.
Treatments that induced <10% relative growth were not considered useful as primary evidence for mutagenicity.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(see % relative growth for each group on the table below)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no, range 7.21-7.68
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: poor
- Precipitation: no
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: yes, preliminary toxicity test

COMPARISON WITH HISTORICAL CONTROL DATA: no, only vehicle controls for assay used

ADDITIONAL INFORMATION ON CYTOTOXICITY: assessed as % relative total growth

Table 1. L5178Y mouse lymphoma data for di-undecyl phthalate (DUP)

Without S-9					With S-9
DUP (µL/mL)	Total mutant colonies	Total viable colonies	% Relative growth	Mut Freq x 10E-6	DUP (µL/mL)	Total mutant colonies	Total viable colonies	% Relative growth	Mut Freq x 10E-6
Acetone	118	732	100	32.2	Acetone	95	667	100	28.5
Acetone	81	633	100	25.6	Acetone	97	588	100	33
Acetone	98	599	100	32.7	Acetone	70	470	100	29.8
Acetone	100	635	100	31.5	Acetone	92	649	100	28.3
2.00	87	642	57.6	27.1	1.00	94	632	43.9	29.7
2.00	68	600	70.7	22.7	1.00	92	690	62.7	26.7
4.00	80	552	30.9	29	2.50	92	530	24.1	34.7
4.00	84	617	30.3	27.2	2.50	104	616	28.4	33.8
5.00	54	468	23.7	23.1	4.00	87	543	20.1	32
5.00	67	456	22.9	29.4	4.00	89	602	28.3	29.6
6.00	87	574	30.4	30.3	5.00	41	390	12	21
6.00	78	589	34.5	26.5	5.00	72	439	13.5	32.8
8.00	89	579	30.7	30.7	5.54	65	313	10.2	41.5
8.00	83	581	30.5	28.6	5.54	41	259	6.1	31.7
10.00	50	395	18.8	25.3	8.00	65	241	4.7	53.9
10.00	46	509	21.1	18.1	8.00	47	255	3.5	36.9
EMS b	1,051	470	39	447.2 a	3-MCA c	446	295	32.2	302.4 a

a Positive result according to the criterion given in the Methods section.

b EMS, ethylmethanesulfonate.

c 3-MCA, 3-methylcholanthrene.

Conclusions:

The test substance was found negative in the Mouse Lymphoma Assay in the presence and absence of metabolic activation.

Executive summary:

DUP was tested in vitro in the L5178Y mouse lymphoma mammalian cell mutation assay in the presence and absence of rat liver S-9 metabolic activation. Mouse lymphoma L5178Y cells were exposed to test material diluted in acetone in RPMI 1640 medium at concentrations of 0.00, 2.00, 4.00, 5.00, 6.00, 8.00 and 10.00 µL/mL (-S9) and 0.00, 1.00, 2.50, 4.00, 5.00, 5.54 and 8.00 µL/mL (+S9). The test was performed in duplicate. Acetone was tested as vehicle control. Positive controls (ethylmethanesulphonate at 0.4 µL/mL without S9 and 3-methylcholanthrene at 4 µg/mL with S9) induced the appropriate response. In the assay with the test substance, no increases in mutant frequency were observed under non-activation or activation conditions. Thus, DUP was determined to be without mutagenic activity in the mouse lymphoma assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium and tryptophan-requiring gene in Escherichia coli

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from Sprague-Dawley male rat livers induced by phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Main test (2 experiments): 0 (solvent control), 156, 313, 625, 1250, 2500 and 5000 μg/plate with and without S9 mix
The top dose was selected based on a preliminary test in which no growth inhibition was observed using doses of 19.5, 78.1, 313 and 1250 μg / plate with and without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO), batch No. K 22063378 534
- Justification for choice of solvent/vehicle: insoluble in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 9-Aminoacridine hydrochloride (ACR) and 2-Aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Preincubation method: In the test tube, 100 μL of test substance solution (or control solvent or positive control), 500 μL of S9 mix (or 0.1 M sodium phosphate buffer solution (pH 7.4) without metabolic activation) and 100 μL of bacterial solution were incubated for 20 minutes at 37°C, under shaking, before adding 2 mL of top agar and pouring onto the surface of a minimal agar plate.
- Cell density at seeding: 4.19-4.44 x 109 cells/mL (TA 1535, WP2 uvrA, TA 98 and TA 100) and 2.03-2.11 x 109 cells/mL (TA 1537) in the 2 experiments.

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours at 37°C

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 2 independent tests with 3 plates/dose/strain.

DETERMINATION OF CYTOTOXICITY
The growth condition of the test strain on the plate was observed using a stereoscopic microscope (× 60) in order to confirm the growth inhibitory action of the test substance on the test strain.

Evaluation criteria:

It was judged as positive when the number of revertant mutant colonies increased almost twice or more as compared with the solvent control and the reproducibility or dependence on the dose of the test substance was recognized.

Statistics:

No statistical methods were used.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 5000 μg/plate in both experiments)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: oil droplets precipitates were observed at a dose of 1250 μg/plate and higher in both experiments with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
Preliminary tests were performed using doses of 19.5, 78.1, 313 and 1250 μg/plate. Growth inhibitory action was not observed in any of the treatment groups with and without metabolic activation. Therefore, in the main test the maximum dose was 5000 μg/plate and 6 doses ( common ratio 2) were set for each test strain with and without S9 metabolic activation.

Table 1: Results of the bacterial reversion test of Ditridecyl phthalate (1st trial) [direct method: -S9]

Compound	Dose  (µg/plate)	Revertant colonies per plate [ Mean ±S. D.]
		TA100	TA1535	WP2 uvrA	TA98	TA1537
DMSO＃	0	120 101 116 [112 ± 10]	12 17 13 [14 ± 3]	22 21 20 [ 21 ± 1]	21 27 23 [ 24 ± 3]	8 9 9 [ 9 ± 1]
Test substance	156	99 115 103 〔106± 8]	18 14 12 [ 15 ± 3]	16 18 20 [ 18土2]	17 21 22 [ 20土3]	13 12 7 [ 11 ± 3]
	313	121 101 101 〔108± 12]	13 17 12 [ 14 ± 3]	20 22 26 [ 23土3]	24 25 19 [ 23 ± 3]	11 14 10 [ 12土2]
	625	100 101 127 [109 ± 15]	18 16 12 [ 15 ± 3]	21 23 21 [ 22 ± 1]	22 15 24 [ 20 ± 5]	12 11 10 [ 11 ± 1]
	1250 +	101 85 112 〔99 ± 14]	14 11 14 [ 13 ± 2]	26 27 18 [ 24 ± 5]	28 20 21 [ 23土4]	9 11 9 [ 10土1]
	2500 +	101 81 102 〔95 ± 12]	12 16 19 〔16 ± 4]	23 20 22 [ 22 ± 2]	21 21 24 〔22 ± 2]	9 8 11 [ 9 ± 2]
	5000 +	97 94 100 [ 97 ± 3]	15 17 16 [ 16 ± 1]	25 27 27 [ 26 ± 1]	21 23 25 [ 23 ± 2]	11* 10* 10* [ 10 ± 1]
Positive control		699 681 694a) [ 691 ± 9]	417 429 398b) [415 ± 16]	183 190 161a) [ 178 ± 15]	561 602 618c) [594 ± 29]	460 505 483d) [483 ± 23]

＃ : Solvent control     *：Growth inhibition was observed

+: Visible precipitation was occured at the end of exposure period

a): AF-2: 2-(2-Furyl)-3-（5-nitro-2-furyl)acrylamide, 0.01 μg/plate, b): NaN3; Sodium azide,

c): AF-2, 0.1 μg/plate d): ACR; 9-Aminoacridine hydrochloride, 80 μ g/plate

Table 2: Results of the bacterial reversion test of Ditridecyl phthalate (1st trial) [activation method : +S9]

Compound	Dose  (µg/plate)	Revertant colonies per plate [ Mean ±S. D.]
		TA100	TA1535	WP2 uvrA	TA98	TA1537
DMSO＃	0	111 109 111 (110土1]	13 18 19 [ 17 ± 3]	2 1 26 27 [ 25土3]	30 25 29 [ 28 ± 3]	13 12 16 〔14 ± 2]
Test substance	156	127 123 102 [117 ± 13]	20 1 3 13 [ 15 ± 4]	20 20 25 [ 22 ± 3]	25 27 28 [ 27 ± 2]	11 14 10 [ 12 ± 2]
	313	109 113 107 [110 ± 3]	17 14 10 [ 14 ± 4]	20 25 21 [ 22 ± 3]	24 23 27 〔25 ± 2]	11 9 11 [ 10 ± 1]
	625	117 114 11 3 [ 115 ± 2]	12 15 15 [ 14 ± 2]	21 24 26 [ 24 ± 3]	37 32 35 [ 35 ± 3]	16 16 17 [ 16 ± 1]
	1250 +	99 127 113 [113 ± 14]	15 15 18 [ 16 ± 2]	26 23 27 〔25 ± 2]	35 32 34 [ 34 ± 2]	12 14 15 [ 14 ± 2]
	2500 +	108 105 1 25 [113 ± 11]	14 16 17 [ 16 ± 2]	27 20 26 [ 24 ± 4]	38 30 37 [ 35 ± 4]	12 10 17 [ 13 ± 4]
	5000 +	121 155 128 [135 ± 18]	18 14 13 〔15 ± 3]	26 29 23 [ 26 ± 3]	34 38 29 [ 34土5]	18 12 13 [ 14土3]
Positive control		703 639 698a) [680 ± 36]	320 314 401b) [345士49]	404 431 414c) [416士14]	347 349 360d) (352 ± 7]	152 171 173b) (165 ± 12]

＃: Solvent control    +: Visible precipitation was occured at the end of exposure period

a) : 2-AA; 2-Aminoanthracene, 1 μg/plate b) : 2-AA, 2 μg/plate c): 2-AA, 10 μg/plate d) : 2-AA, 0.5 μg/plate

Table 3: Results of the bacterial reversion test of Ditridecyl phthalate (2nd trial) [direct method: -S9]

Compound	Dose  (µg/plate)	Revertant colonies per plate [ Mean ±S. D.]
		TA100	TA1535	WP2 uvrA	TA98	TA1537
DMSO＃	0	120 111 120 (117土5]	16 15 18 [ 16 ± 2]	24 25 23 [ 24土1]	24 24 23 [ 24 ± 1]	8 5 5 〔6 ± 2]
Test substance	156	114 127 131 [124 ± 9]	19 20 16 [ 18 ± 2]	26 23 22 [ 24 ± 2]	33 29 22 [ 28 ± 6]	6 8 6 [ 7 ± 1]
	313	124 133 111 [123 ± 11]	15 12 16 [ 14 ± 2]	21 25 22 [ 23 ± 2]	28 27 30 〔28 ± 2]	5 5 7 [6 ± 1]
	625	133 128 126 [ 129 ± 4]	19 18 17 [ 18 ± 1]	22 23 19 [ 21 ± 2]	22 28 28 [ 26 ± 3]	5 4 5 [5 ± 1]
	1250 +	116 115 132 [121 ± 10]	14 14 18 [ 15 ± 2]	25 19 22 〔22 ± 3]	25 22 18 [ 22 ± 4]	5 3 3 [ 4 ± 1]
	2500 +	121 105 112 [113 ± 8]	21 15 16 [ 17 ± 3]	18 26 21 [ 22 ± 4]	21 33 32 [ 29 ± 7]	4 5 4 [ 4 ± 1]
	5000 +	121 155 128 [135 ± 18]	18 20 18 〔19 ± 1]	19 27 27 [ 24 ± 5]	27 33 36 [ 32土5]	7* 9* 5* [7土2]

＃: Solvent control     *：Growth inhibition was observed

+: Visible precipitation was occured at the end of exposure period

a): AF-2: 2-(2-Furyl)-3-（5-nitro-2-furyl)acrylamide, 0.01 μg/plate, b): NaN3; Sodium azide,

c): AF-2, 0.1 μg/plate d): ACR; 9-Aminoacridine hydrochloride, 80 μ g/plate

Table 4: Results of the bacterial reversion test of Ditridecyl phthalate (2nd trial) [activation method : +S9]

Compound	Dose  (µg/plate)	Revertant colonies per plate [ Mean ±S. D.]
		TA100	TA1535	WP2 uvrA	TA98	TA1537
DMSO＃	0	119 114 133 [122 ± 10]	13 14 17 [ 15 ± 2]	30 30 24 [ 28 ± 3]	30 32 32 [ 31 ± 1]	10 13 9 [ 11 ± 2]
Test substance	156	124 115 107 [ 115 ± 9]	13 18 11 [ 14 ± 4]	33 26 25 [ 28 ± 4]	33 26 25 [ 28 ± 4]	9 14 13 [12 ± 3]
	313	115 118 131 [ 121 ± 9]	20 18 17 [ 18 ± 2]	33 26 34 [ 31 ± 4]	33 26 34 [ 31 ± 4]	10 12 12 [ 11 ± 1]
	625	109 129 127 [122 ± 11]	20 18 13 [ 17 ± 4]	23 26 32 [ 27 ± 5]	23 26 32 [ 27 ± 5]	10 7 11 [ 9 ± 2]
	1250 +	133 121 131 [ 128 ± 6]	16 17 17 [ 17 ± 1]	23 30 35 [ 29 ± 6]	23 30 35 [ 29 ± 6]	12 9 16 [ 12 ± 4]
	2500 +	130 128 121 [ 126 ± 5]	15 18 17 [ 17 ± 2]	41 29 33 [ 34 ± 6]	41 29 33 [ 34 ± 6]	13 13 11 [ 12土l]
	5000 +	125 122 120 〔122土3]	21 17 19 [ 19 ± 2]	38 39 42 [ 40 ± 2]	38 39 42 [ 40 ± 2]	17 13 12 [ 14 ± 3]
Positive control		551 492 565a) [536 ± 39]	336 364 418b) [ 373 ± 42	348 333 368d) [350 ± 18]	348 333 368d) [350 ± 18]	151 153 141b) [ 148 ± 6]

＃: Solvent control    +: Visible precipitation was occured at the end of exposure period

a) : 2-AA; 2-Aminoanthracene, 1 μg/plate b) : 2-AA, 2 μg/plate c): 2-AA, 10 μg/plate d) : 2-AA, 0.5 μg/plate

Conclusions:

Ditridecyl phthalate was not mutagenic with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA with and without metabolic activation.

Executive summary:

In a bacterial reverse mutation assay following a method according to OECD guideline 471 and GLP, the test item ditridecyl phthalate diluted in Dimethylsulfoxide (DMSO) was tested with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA with and without metabolic activation (S9) using the preincubation method. Based on results of a preliminary assay, for the main test two independent experiments and 3 replicates of each were conducted with doses of 0 (solvent control), 156, 313, 625, 1250, 2500 and 5000 μg/plate with and without S9 mix. Growth inhibition was only found at 5000 μg/plate in the strain TA1537 without S9 mix. Oil droplets precipitates were observed at a dose of 1250 μg/plate and up to the highest dose with and without metabolic activation. The positive controls induced more than twice the revertant colonies induced in the the solvent control. Finally, the test item did not show mutagenic activity in any of the bacterial strains tested and thus, it is considered a non-mutagenic in the bacterial reverse mutation test.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Consistent with recommended guidelines for studies of this type and also in accordance with international standards.

Qualifier:

according to guideline

Guideline:

other: recommended guidelines for studies of this type (Dean B., 1984 and Galloway S., 1985) and with international standards (Richardson C., 1989).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Remarks:

This information was not reported

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Source: no info
- Type and identity of media: McCoy’s 5A medium containing 10% fetal bovine serum and 2 mM L-glutamine at ca. 37°C in 4–6% CO2 in air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from Aroclor-induced Sprague-Dawley rat liver.

Test concentrations with justification for top dose:

0 (solvent control), 5, 10, 20, 40, 80 and 160 μg/mL.
The test concentrations were based on preliminary testing, which revealed precipitation and debris at 160 μg/mL both in the presence and absence of metabolic activation. Based on visual inspection it appeared that the limit of solubility was in the range of 20– 40 μg/mL.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone (50 μL)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 7,12-dimethyl (benz[a]anthracene (DMBA) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium.
- Cell density at seeding (if applicable): 1.2 x 106 cells (20-h harvest) and 0.8 x 106 cells (44-h harvest) ca. 24 h prior to test material administration.

DURATION
- Exposure duration: 3 h (+/-S9 initial assay); 3h (+S9 repeat assay) and 20 h (-S9 repeat assay)
- Harvest times: 20 h or 44 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Not specified.

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not specified.

NUMBER OF CELLS EVALUATED: 200

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 (100 per flask)

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index evaluated based on 1000 cells scored.

Evaluation criteria:

Treated groups were compared with a control by the Fisher exact test, and differences were considered significant at P ≤ 0.05.

Statistics:

Statistical evaluation followed the recommendations of Richardson C., 1989.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1. Clastogenic response of DINP (68515–48–0) in the in vitro chromosomal aberration assay in Chinese Hamster ovary cells utilizing S9 derived from livers of Aroclor-induced Sprague-Dawley rats a

Test material and dose (µg ml-1)	Percent mitotic cells b			% Aberrant cells c
	Initial	Repeat		Initial	Repeat
	20-h	20-h	44-h	20-h	20-h	44-h
Activated (+S9)
Acetone d	8.5	8.9	4.0	2.0	1.5	3.5
DINP 40	6.9	7.9	5.2	3.5	2.0	3.5
DINP 80	7.0	9.1	6.2	3.0	1.0	3.0
DINP 160	10.2	10.8	4.5	3.0	2.5	5.0
DMBA 10	1.2	1.4	–	65.0**	63.0**	–
Non-activated (-S9)
Acetone d	11.2	5.5	5.1	1.0	1.0	2.0
DINP 40	8.3	3.7	5.3	2.5	2.5	1.5
DINP 80	7.8	4.1	4.2	2.0	1.0	2.5
DINP 160	5.5	3.0	4.7	2.0	4.0	0.5
MNNG e (0.6)	3.7	3.2	–	10.5**	8.0**	–

a Based on evaluation of 200 cells except for 7,12-dimethyl (benz[a]anthracene (DMBA), which is based on 100 cells (combination of data from two flasks).

b % Mitotic cells = number of mitotic cells per 1000 total cells.

c % Aberrant cells = number of cells with aberrations / total number of cells scored  x 100.

d Vehicle control group. Dose = 50 µl per flask.

e MNNG = N-methyl-N-nitro-N-nitrosoguanidine.

**Statistically significantly higher than vehicle control (P < 0.01).

Conclusions:

DINP was found negative in an in vitro chromosomal aberration test in CHO cells.

Executive summary:

In an in vitro chromosomal aberration test conducted according to recommended guidelines for studies of this type (Dean B., 1984 and Galloway S., 1985) and with international standards (Richardson C., 1989), Chinese hamster Ovary (CHO) cells were exposed to DINP in McCoys 5A medium with and without metabolic activation (S9 fraction of livers from Aroclor 1254-induced Sprague-Dawley rats) at concentrations of 0 (solvent control with acetone), 5, 10, 20, 40, 80 and 160 μg/mL. 7,12-dimethyl (benz[a]anthracene (DMBA) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were used as positive controls with and without metabolic activation respectively. Frequencies of chromosomal aberrations were similar in treated and control groups at all concentrations and collection times in both the presence and absence of metabolic activation. Significant increases in chromosomal aberrations were induced by the positive controls. Similar results were obtained in a repeat assay, confirming the validity of the findings. Thus, DINP is not considered as cytogenetic.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Both the target substance (DUP) and the source substance (DTP) belong to the OECD HPV category High Molecular Weight Phthalate Ester (HMWPE) which consists of esters with an alkyl carbon backbone with 7 carbon (C) atoms or greater. Thus, these substances share the same functional groups and also have comparable environmental and toxicological properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at highest dose tested)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: read-across from an analogue determined to be not mutagenic in bacteria

Conclusions:

Based on the read-across approach from the analogue substante Ditridecyl phthalate (DTP), diundecyl phthalate (DUP) was determined to be not mutagenic with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA with and without metabolic activation.

Executive summary:

In a bacterial reverse mutation assay following a method according to OECD guideline 471 and GLP, the test item ditridecyl phthalate diluted in Dimethylsulfoxide (DMSO) was tested with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA with and without metabolic activation (S9) using the preincubation method. Based on results of a preliminary assay, for the main test two independent experiments and 3 replicates of each were conducted with doses of 0 (solvent control), 156, 313, 625, 1250, 2500 and 5000 μg/plate with and without S9 mix. Growth inhibition was only found at 5000 μg/plate in the strain TA1537 without S9 mix. Oil droplets precipitates were observed at a dose of 1250 μg/plate and up to the highest dose with and without metabolic activation. The positive controls induced more than twice the revertant colonies induced in the the solvent control. Finally, the test item did not show mutagenic activity in any of the bacterial strains tested and thus, it is considered a non-mutagenic in the bacterial reverse mutation test. Based on these results, the read-across approach was applied and diundecyl phthalate (DUP) was determined to be not mutagenic.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance (DUP) belongs to the OECD HPV category High Molecular Weight Phthalate Ester (HMWPE) which consists of esters with an alkyl carbon backbone with 7 carbon (C) atoms or greater. The source substance (DINP) although is not formally part of this category as it was assessed previously, it satisfies the category definition as its backbone length is C7 or above and also produces similar effects of developmental or reproductive toxicity. Thus, these substances share the same functional groups and also have comparable environmental and toxicological properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: read-across from an analogue determined to be not mutagenic in a chromosomal aberration test

Conclusions:

Based on the read-across approach from the analogue diisononyl phthalate (DINP) assessed in an vitro chromosomal aberration test, diundecyl phthalate (DUP) was determined to be non-cytogenic to mammalian cells.

Executive summary:

In an in vitro chromosomal aberration test conducted according to recommended guidelines for studies of this type (Dean B., 1984 and Galloway S., 1985) and with international standards (Richardson C., 1989), Chinese hamster Ovary (CHO) cells were exposed to DINP in McCoys 5A medium with and without metabolic activation (S9 fraction of livers from Aroclor 1254-induced Sprague-Dawley rats) at concentrations of 0 (solvent control with acetone), 5, 10, 20, 40, 80 and 160 μg/mL. 7,12-dimethyl (benz[a]anthracene (DMBA) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were used as positive controls with and without metabolic activation respectively. Frequencies of chromosomal aberrations were similar in treated and control groups at all concentrations and collection times in both the presence and absence of metabolic activation. Significant increases in chromosomal aberrations were induced by the positive controls. Similar results were obtained in a repeat assay, confirming the validity of the findings. Thus, DINP is not considered as cytogenetic. Based on these results, the read-across was applied and diundecyl phthalate (DUP) was determined to be non-cytogenic to mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In-vivo micronucleus assay: Key studies: Read-across approach. Test method OECD 474. Based on the read-across approach from the analogues diisononyl phthalate (DINP) and diisodecyl phthalate (DIDP) assessed in the mammalian erythrocyte micronucleus test, DUP was determined to be non-cytogenic to mammalian cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

(this information was not provided)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 6-9 weeks
- Weight at study initiation: 17-35 g
- Housing: husbandry was consistent with the recommendations of the National Institute of Health (NIH), 1985.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No information

Duration of treatment / exposure:

single dose 1 day

Frequency of treatment:

Once

Post exposure period:

24, 48 and 72 hours (killing times)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

(vehicle control)

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide. 5 males and 5 females. Killing time: 24 h post administration
- Route of administration: oral, gavage
- Doses / concentrations: 20 mg/kg

Tissues and cell types examined:

Bone marrow erythrocytes cells (from both femora)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range-finding study was used to select doses for the definitive test, but, because there was no evidence of toxicity, the highest dose was 5 g/kg based on TSCA (Toxic Substances Control Act) guidelines (US EPA 798.5395).

TREATMENT AND SAMPLING TIMES:
Both femurs were removed from each treated mouse and the proximal ends were cut to expose the bone marrow, which was then aspirated with fetal bovine serum into a centrifuge tube. The cells were collected by centrifugation and slides were prepared.
DETAILS OF SLIDE PREPARATION:
After fixation in methanol, the slides were stained with acridine orange for ca. 1–2 min.

METHOD OF ANALYSIS:
Slides were evaluated at 400 X by fluorescence microscopy.
A total of 1000 erythrocytes were counted from each animal, and the total numbers of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. A total of 1000 PCEs were evaluated for the presence of micronuclei.

Statistics:

Statistical analysis included means and standard deviations of the micronucleus test data, and a test of equality of means was performed by standard one-way analysis of variance (Snedecor G., 1989).
Because the only statistically significant differences were between the positive and negative control groups, no additional statistical analysis was carried out.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
There was no evidence of toxicity up to the highest dose tested.

RESULTS OF DEFINITIVE STUDY
- Mortality: All animals survived.
- Signs of toxicity: there was no evidence of toxicity.
- Induction of micronuclei (for Micronucleus assay): There were no significant differences between treatment groups and controls for either male or female mice at any dose or collection time.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE/NCE remained unaffected by the treatment and was statistically not different from the control values.
- Positive control: Treatment with cyclophosphamide, the positive control, induced a significant increase in micronuclei in both males and females, confirming the adequacy of the testing procedure.

Table 1. In vivo micronucleus evaluation of DIDP (68515–49–1) in male CD-1 mice a

Treatment	Dose	Harvest time (h)	% Micronucleated PCEs (1000 per animal)			Ratio PCE/NCE mean ± S.E.
			Males	Females	Total	Males	Females
Vehicle control (corn oil)	10 ml kg-1	24	0.06 ± 0.04	0.04 ± 0.02	0.05 ± 0.02	0.67 ± 0.31	0.58 ± 0.18
		48	0.00 ± 0.00	0.04 ± 0.02	0.02 ± 0.01	0.59 ± 0.08	1.00 ± 0.08
		72	0.08 ± 0.04	0.02 ± 0.02	0.05 ± 0.02	0.89 ± 0.17	1.09 ± 0.13
Positive control (cyclophosphamide)		24	2.04 ± 0.34*	1.76 ± 0.28*	1.90 ± 0.21*	0.50 ± 0.12	0.48 ± 0.08
DIDP dose	1250 mg kg-1	24	0.16 ± 0.04	0.10 ± 0.05	0.13 ± 0.03	0.35 ± 0.08	0.97 ± 0.09
		48	0.06 ± 0.02	0.06 ± 0.04	0.06 ± 0.02	0.69 ± 0.14	1.15 ± 0.10
		72	0.02 ± 0.02	0.10 ± 0.04	0.06 ± 0.03	0.79 ± 0.15	1.10 ± 0.14
	2500 mg kg-1	24	0.08 ± 0.04	0.08 ± 0.04	0.08 ± 0.02	0.49 ± 0.08	0.83 ± 0.19
		48	0.02 ± 0.02	0.06 ± 0.02	0.04 ± 0.02	0.91 ± 0.16	1.00 ± 0.15
		72	0.04 ± 0.02	0.00 ± 0.00	0.02 ± 0.01	0.62 ± 0.16	0.96 ± 0.11
	5000 mg kg-1	24	0.08 ± 0.04	0.06 ± 0.02	0.07 ± 0.02	0.38 ± 0.05	1.05 ± 0.17
		48	0.00 ± 0.00	0.02 ± 0.02	0.01 ± 0.01	0.90 ± 0.10	1.02 ± 0.17
		72	0.02 ± 0.02	0.06 ± 0.04	0.04 ± 0.02	0.89 ± 0.03	1.27 ± 0.19

a Values are means ± SEM; *significantly greater than the corresponding vehicle control (P < 0.05).

PCE = polychromatic erythrocyte; NCE = normochromatic erythrocyte

Conclusions:

DIDP was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.

Executive summary:

An in-vivo micronucleus test was performed with Di(isodecyl) Phthalate (DIDP) according to OECD guideline 474 and EU method B.12. Five mice per sex and per group were exposed once (single dose) to test item diluted in corn oil at doses of 0 (vehicle control), 1250, 2500 and 5000 mg/kg bw, based on preliminary results. Animals were killed 24, 48 and 72 hours after administration of the test compound. Cyclophosphamide was used as a positive control (5 mice per sex at 20 mg/kg and killed 24 h post treatment). For each animal, bone marrow was extracted from both femora and the slides were prepared for erythrocyte micronuclei observation. There were no significant differences between treatment groups and controls for either male or female mice at any dose or collection time. The ratio of PCE/NCE remained unaffected by the treatment and was statistically not different from the control values. Based on these results, DIDP was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

(this information was not provided)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 6-9 weeks
- Weight at study initiation: 17-35 g
- Housing: husbandry was consistent with the recommendations of the National Institute of Health (NIH), 1985.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: not specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No information

Duration of treatment / exposure:

2 consecutive days

Frequency of treatment:

once a day

Post exposure period:

Animals were sacrificed on 3rd day

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

(vehicle control)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide. 5 males administered 2 consecutive days and sacrificed on 3rd day
- Route of administration: oral, gavage
- Doses / concentrations: 20 mg/kg bw /day

Tissues and cell types examined:

Bone marrow erythrocytes cells (from both femora)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range-finding study was used to select doses for the definitive test, but, because there was no evidence of toxicity, the highest dose was 2 g/kg/day based on OECD guideline 474 recommendations.

TREATMENT AND SAMPLING TIMES:
Both femurs were removed from each treated mouse and the proximal ends were cut to expose the bone marrow, which was then aspirated with fetal bovine serum into a centrifuge tube. The cells were collected by centrifugation and slides were prepared.
DETAILS OF SLIDE PREPARATION:
After fixation in methanol, the slides were stained with acridine orange for ca. 1–2 min.

METHOD OF ANALYSIS:
Slides were evaluated at 400 X by fluorescence microscopy.
A total of 1000 erythrocytes were counted from each animal, and the total numbers of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. A total of 1000 PCEs were evaluated for the presence of micronuclei.

Statistics:

Statistical analysis included means and standard deviations of the micronucleus test data, and a test of equality of means was performed by standard one-way analysis of variance (Snedecor G., 1989).
Because the only statistically significant differences were between the positive and negative control groups, no additional statistical analysis was carried out.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
There was no evidence of toxicity up to the highest dose tested. Since there were no differences between males and females, the definitive test utilized male mice only.

RESULTS OF DEFINITIVE STUDY
- Mortality: All animals survived.
- Signs of toxicity: there was no evidence of toxicity.
- Induction of micronuclei (for Micronucleus assay): Frequency of micronucleus formation was not elevated at any dose. The micronucleus frequency at 500 mg/kg DINP was significantly below control values and assumed to be unimportant toxicologically.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE/NCE remained unaffected by the treatment and was statistically not different from the control values.
- Positive control: Treatment with cyclophosphamide, the positive control, induced a significant increase in micronucleus formation, confirming the sensitivity of the testing procedure.

Table 1. In vivo micronucleus evaluation of DINP (68515–48–0) in male CD-1 mice a

Dose group	% Polychromatic  erythrocytes of cells scored	% Polychromatic  erythrocytes with micronuclei
Vehicle control	50.3 ± 5.0	0.22 ± 0.04
500 mg / kg DINP	51.2 ± 3.0	0.14 ± 0.07*
1000 mg / kg DINP	50.9 ± 6.2	0.18 ± 0.03
2000 mg / kg DINP	51.6 ± 4.5	0.25 ± 0.05
20 mg / kg cyclophosphamide (positive control)	41.7 ± 7.6**	2.20 ± 0.48**

a Each group contained five male mice; *significant at P < 0.05; **significant at P < 0.01.

Conclusions:

DINP was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.

Executive summary:

An in-vivo micronucleus test was performed with Di(isononyl) Phthalate (DINP) according to OECD guideline 474 and EU method B.12. Five male mice per group were exposed to test item on 2 consecutive days at doses of 0 (vehicle control), 500, 1000 and 2000 mg/kg bw /day, based on preliminary results. Cyclophosphamide was used as a positive control (5 male mice at 20 mg/kg/day). Animals were killed at third day. For each animal, bone marrow was extracted from both femora and the slides were prepared for erythrocyte micronuclei observation. Frequency of micronucleus formation was not elevated at any dose and there was no evidence of toxicity. The ratio of PCE/NCE remained unaffected by the treatment and was statistically not different from the control values. Based on these results, DINP was determined to be not mutagenic in the mammalian erythrocyte micronucleus test.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance (DUP) belongs to the OECD HPV category High Molecular Weight Phthalate Ester (HMWPE) which consists of esters with an alkyl carbon backbone with 7 carbon (C) atoms or greater. The source substance (DIDP) although is not formally part of this category as it was assessed previously, it satisfies the category definition as its backbone length is C7 or above and also produces similar effects of developmental or reproductive toxicity. Thus, these substances share the same functional groups and also have comparable environmental and toxicological properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: read-across from an analogue determined to be not mutagenic in the mammalian erythrocyte micronucleus test

Conclusions:

Based on the read-across approach from the analogue diisodecyl phthalate (DIDP) assessed in the mammalian erythrocyte micronucleus test, diundecyl phthalate (DUP) was determined to be non-cytogenic to mammalian cells.

Executive summary:

An in-vivo micronucleus test was performed with Di(isodecyl) Phthalate (DIDP) according to OECD guideline 474 and EU method B.12. Five mice per sex and per group were exposed once (single dose) to test item diluted in corn oil at doses of 0 (vehicle control), 1250, 2500 and 5000 mg/kg bw, based on preliminary results. Animals were killed 24, 48 and 72 hours after administration of the test compound. Cyclophosphamide was used as a positive control (5 mice per sex at 20 mg/kg and killed 24 h post treatment). For each animal, bone marrow was extracted from both femora and the slides were prepared for erythrocyte micronuclei observation. There were no significant differences between treatment groups and controls for either male or female mice at any dose or collection time. The ratio of PCE/NCE remained unaffected by the treatment and was statistically not different from the control values. Based on these results, DIDP was determined to be not mutagenic in the mammalian erythrocyte micronucleus test. Based on these results, the read-across was applied and diundecyl phthalate (DUP) was determined to be non-cytogenic to mammalian cells.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance (DUP) belongs to the OECD HPV category High Molecular Weight Phthalate Ester (HMWPE) which consists of esters with an alkyl carbon backbone with 7 carbon (C) atoms or greater. The source substance (DINP) although is not formally part of this category as it was assessed previously, it satisfies the category definition as its backbone length is C7 or above and also produces similar effects of developmental or reproductive toxicity. Thus, these substances share the same functional groups and also have comparable environmental and toxicological properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: read-across from an analogue determined to be not mutagenic in the mammalian erythrocyte micronucleus test

Conclusions:

Based on the read-across approach from the analogue diisononyl phthalate (DINP) assessed in the mammalian erythrocyte micronucleus test, diundecyl phthalate (DUP) was determined to be non-cytogenic to mammalian cells.

Executive summary:

An in-vivo micronucleus test was performed with Di(isononyl) Phthalate (DINP) according to OECD guideline 474 and EU method B.12. Five male mice per group were exposed to test item on 2 consecutive days at doses of 0 (vehicle control), 500, 1000 and 2000 mg/kg bw /day, based on preliminary results. Cyclophosphamide was used as a positive control (5 male mice at 20 mg/kg/day). Animals were killed at third day. For each animal, bone marrow was extracted from both femora and the slides were prepared for erythrocyte micronuclei observation. Frequency of micronucleus formation was not elevated at any dose and there was no evidence of toxicity. The ratio of PCE/NCE remained unaffected by the treatment and was statistically not different from the control values. Based on these results, DINP was determined to be not mutagenic in the mammalian erythrocyte micronucleus test. Based on these results, the read-across was applied and diundecyl phthalate (DUP) was determined to be non-cytogenic to mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Key studies from experimental results with the test substance and some analogues:

In vitro gene mutation in bacteria:

Diundecyl phthalate (DUP): In a bacterial reverse mutation assay following a method similar to OECD guideline 471, the test item diundecyl phthalate diluted in Dimethylsulfoxide (DMSO) was found negative in Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 with and without metabolic activation (S9) using the preincubation method.

Ditridecyl phthalate (DTP): In a bacterial reverse mutation assay following a method according to OECD guideline 471 and GLP, the test item ditridecyl phthalate diluted in Dimethylsulfoxide (DMSO) was found negative in Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA with and without metabolic activation (S9) using the preincubation method. Based on these results, the read-across approach was applied and diundecyl phthalate (DUP) was determined to be not mutagenic in these tester strains.

In vitro mammalian chromosome aberration:

Diisononyl phthalate (DINP): In an in vitro chromosomal aberration test conducted according to recommended guidelines for studies of this type (Dean B., 1984 and Galloway S., 1985) and with international standards (Richardson C., 1989), Chinese hamster Ovary (CHO) cells were exposed to DINP in McCoys 5A medium with and without metabolic activation (S9 fraction of livers from Aroclor 1254-induced Sprague-Dawley rats) at concentrations of 0 (solvent control with acetone), 5, 10, 20, 40, 80 and 160 μg/mL. DINP was not found as cytogenetic. Based on these results, the read-across was applied and diundecyl phthalate (DUP) was determined to be non-cytogenic to mammalian cells.

In vitro gene mutation study in mammalian cells:

Diundecyl phthalate (DUP): DUP was tested in vitro in the L5178Y mouse lymphoma mammalian cell mutation assay in the presence and absence of rat liver S-9 metabolic activation. Mouse lymphoma L5178Y cells were exposed to test material diluted in acetone in RPMI 1640 medium at concentrations of 0.00, 2.00, 4.00, 5.00, 6.00, 8.00 and 10.00 µL/mL (-S9) and 0.00, 1.00, 2.50, 4.00, 5.00, 5.54 and 8.00 µL/mL (+S9). DUP was determined to be without mutagenic activity in the mouse lymphoma assay.

In-vivo genetic toxicity:

Diisononyl phthalate (DINP): An in-vivo micronucleus test was performed with DINP according to OECD guideline 474 and EU method B.12. Five male mice per group were exposed to test item on 2 consecutive days at doses of 0 (vehicle control), 500, 1000 and 2000 mg/kg bw /day, based on preliminary results. Frequency of micronucleus formation was not elevated at any dose and there was no evidence of toxicity. The ratio of PCE/NCE remained unaffected by the treatment and was statistically not different from the control values. Based on these results, DINP was determined to be not mutagenic in the mammalian erythrocyte micronucleus test. Based on these results, the read-across was applied and diundecyl phthalate (DUP) was determined to be non-cytogenic to mammalian cells.

Di(isodecyl) Phthalate (DIDP): An in-vivo micronucleus test was performed with DIDP according to OECD guideline 474 and EU method B.12. Five mice per sex and per group were exposed once (single dose) to test item diluted in corn oil at doses of 0 (vehicle control), 1250, 2500 and 5000 mg/kg bw, based on preliminary results. There were no significant differences between treatment groups and controls for either male or female mice at any dose or collection time. The ratio of PCE/NCE remained unaffected by the treatment and was statistically not different from the control values. Based on these results, DIDP was determined to be not mutagenic in the mammalian erythrocyte micronucleus test. Based on these results, the read-across was applied and diundecyl phthalate (DUP) was determined to be non-cytogenic to mammalian cells.

Justification for classification or non-classification

Based on the available information, the substance is considered to be negative for genetic toxicity, and therefore the substance is not classified in accordance with CLP Regulation (EC) no 1272/2008.