COASOL 290 PLUS

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity (Bacterial and Mammalian):

There are no data available for the dibutyl esters, however studies are available for dibutyl adipate, dimethyl glutarate and also the mix of methyl esters. In the available bacterial mutagenicity studies all test materials were negative for mutagenicity in the presence and absence of metabolic activation. The results are consistent across the group of substances and therefore it can be concluded that the mix of dibutyl adipate and dibutyl glutarate would be negative for bacterial mutagenicity.

Chromosomal aberration:

Data are available for dibutyl adipate, the mix of methyl esters, and dimethyl glutarate. In the in vitro studies (dibutyl adipate and mix of methyl esters) the test materials were negative in the absence of metabolic activation, but weakly positive in the presence of metabolic activation. This clastogenicity in vitro appears to be common to the dibasic esters when tested in the presence of metabolic activation. This positive result appears to be inconsistent with what is known about the metabolites of the dibasic esters (the acids and the alcohols). The first step in metabolism of these compounds is the hydrolysis, releasing the acids and alcohols. The genotoxicity data on these substances indicates an absence of clastogenicity, in fact succinate, glutarate and adipate are all present endogenously and so are unlikely to possess clastogenic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

14-May-2001 to 11-Feb-2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The members of the category are all alcohol esters of dicarboxylic acids. All category members are manufactured by reacting an alcohol (methanol, butanol or isobutanol) with single dicarboxylic acids, succinic, glutaric or adipic acids or mixtures of these acids. The ester bonds are effectively metabolised by the body releasing the component alcohols and acids. The difference between members involves 3 parameters: 1) the alcohol used to esterify the acids, 2) the length of the acid molecule (4C, 5C or 6C) and 3) the presence of individual esters or mixtures thereof.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
The toxicity profile of the members (ecotoxicity and human health toxicity and the environmental fate) is consistent. All have low acute toxicity potential, are not sensitising, are mildly irritating to eyes and upper respiratory tract (where vapour pressure allows exposure), are not genotoxic or clastogenic (in vivo) and have minimal systemic toxicity. Data are available predominantly for the methyl esters (individual and mixture), dibutyl adipate and diisobutyl esters (mixture). Within the category, read across is used to cover the higher tier human health toxicity studies predominantly.

See attached document with the justification for the category/read-across approach.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Preliminary toxicity test: 78-5000 ug/L (halving concentrations)
-S9: 315-5000 ug/L (5 halving concentrations) in Test 1 and Test 2
+S9: 400, 500, 600, 700, 800, and 900 ug/L in Test 1
+S9: 600, 800, 900, 1000, 1100 and 1200 ug/L 9 in Test 2

Vehicle / solvent:

DMSO

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Positive control in absence of S9

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

Positive contol in presence of S9

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Details on test system and experimental conditions:

Dimethyl glutarate was evaluated for its mutagenic potential in Chinese hamster ovary cells. The test system evaluated the potential of DMG to induce a forward mutation at the functionally hemizygous hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. A prelimiary toxicity test was performed on cell suspensions in nutrient medium in presence and absence of S9. Cells were incubated for 20 hours at 37C in a humid atmosphere fo 5% CO2 in the air prior to exposure to the test substance on Day 1. Two mL of S9 or nutirent medium were added to the cell suspension followed by 120 ul of the test material or control solvent. The treated cell suspension were returned to the incubator for an additional 4 hour. Cell suspension were covered during incubation to prevent loss of test material.

At the end of treatment, the cells were harvested, washed and seeded on 3 60 mm dishes (200 cells per dish) with nutrient medium. The plates were incubated for at least 7 days, then growing colonies were fixed stained and counted. Cell survival was expressed as the plating efficiency relative to solvent controls. Concentrations for the main test were chosen based on these results.

For the main test, cell suspensions were prepared as described above except that duplicate cultures were used for each test concentration and positive control and quadruplicate culture for solvent controls. At least 5 serial dilutions were used at concentrations expected to span LC80 to LC0. Following exposure to test substance for 4 hours and plating as described above, 10x6 cells were seeded in a flask and incubated for 7 days to allow expression of the mutant phenotype. The cultures were subcultured on days 4 or 5 and after a total of 7 days were harvested by trypsinization (Day 8). the cells were harvested, washed and seeded on 3 60 mm dishes (200 cells per dish) with nutrient medium. The plates were incubated for at least 7 days, then growing colonies were fixed stained and counted. Cell survival was expressed as the plating efficiency relative to solvent controls. Concentrations for the main test were chosen based on these results.

Evaluation criteria:

Cytotoxicity - [Total colonies on plates (treated)/total conlonies on plates (untreated)] x 100

Plating efficiency (PE)- [Total number of viable colonies for each treated group/number of plates scored for colony formation x 200] x 100

Mutation frequency (MF) - [Total number of mutant colonies x 5*/PE x number ofuncontaminated plates] x 100

*5 represents a correction factor for the number of uncontaminated plates which normally equals 5, but may have been less

Criteria for a positive response were:
1) Demonstration of a statistically significant increase in mutation frequency
2) Evidence of a dose-relationship over at least two dose-levels
3) Demonstration of reproduibility in any increases in mutant frequency
4) Mean mutation frequency should fall outside the upper limit of historical control range of 20 mutants per million survivors with acorresponding survival rate of 20% or greater

Species / strain:

Chinese hamster Ovary (CHO)

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

Test 1: Dimethyl Glutarate in the absence of S9

Concentration	Mean Relative Survival Day 1	Mean Relative Survival Day8	Mean Mutation Frequency
0	100	100	7.47
313	119	100	9.34
625	120	109	2.21
1250	93	94	6.92
2500	81	96	4.58
5000	78	95	8.60
EMS ¿Positive control	99	80	392.67*

*p<0.001

Test 2: Dimethyl Glutarate in the absence of S9

Concentration	Mean Relative Survival Day 1	Mean Relative Survival Day8	Mean Mutation Frequency
0	100	100	11.35
313	105	98	12.69
625	94	102	9.81
1250	104	99	11.88
2500	97	95	7.9
5000	83	102	20.06
EMS ¿Positive control	76	94	449.50*

*p<0.001

Test 1: Dimethyl Glutarate in the presence of S9

Concentration	Mean Relative Survival Day 1	Mean Relative Survival Day8	Mean Mutation Frequency
0	100	100	10.51
400	100	96	2.89
500	88	92	8.79
600	82	92	2.96
700	82	98	2.84
800	29	94	4.09
900	82	97	9.59
3-MC- positive control	100	94	202.53*

*p<0.001

Conclusions:

Dimethyl glutarate did not demonstrate mutagenic potential in the in vitro HPRT cell mutation assay in the presence and absence of S9 metabolic activation.

Executive summary:

Dimethyl glutarate was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation at the functionally hemizygous hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells.

The test was repeated more than twice, but several tests, particularly in the presence of S9 mix, were rejected, because there were insufficient mutagenicity data to claim a valid test. The results of four independent tests, two in the absence of exogenous metabolic activation (S9 mix), and two in the presence of S9 mix are reported.

In the preliminary toxicity test, cultures were exposed to 7 halving concentrations ranging from 78- 5000 µg/ml. In the absence of S9 mix, cultures were exposed to 5 halving concentrations ranging from 313-5000 µg/ml in both mutagenicity tests. In the presence of S9 mix, cultures were exposed to 400, 500, 600, 700, 800 or 900 µg/ml in the first mutagenicity test, and to 600, 800, 900, 1000, 1100 and 1200 µg/ml in the second. In all tests exposure was for 4 hours.

The preliminary toxicity test showed Day 1 relative cell survival of 65% after exposure to 5000 µg/ml in the absence of S9 mix and no survival after exposure to 1250 µg/ml or higher concentrations in the presence of S9 mix. In the main mutagenicity tests, slight toxicity was observed after exposure to dimethyl glutarate in tests in the absence of S9 mix, so that Day 1 relative cell survival was reduced to 78% or 83% by 5000 µg/ml in the first and second main test respectively. In the presence of S9 mix, Day1 relative cell survival was reduced to 82% by 900 µg/ml in the first main test, and to 27% by 1200 µg/ml in the second.

No significant increases in mutant frequency were observed in any of the tests either in the absence or presence of S9 mix, nor was there evidence of a concentration-response relationship. In all tests the positive control substances increased mutant frequencies significantly (P<0.001)

In the presence of S9 mix, dimethyl glutarate failed to reduce the Day1 cell survival to 10-20% of solvent control values, but it is considered that a sufficient number of high concentrations in a toxic range were tested to assess mutagenic potential adequately.

It was concluded that dimethyl glutarate did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay in either the absence or presence of S9 metabolic activation, under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: only 2 of 4 recommended strains (TA98, TA100) were used in this study

Principles of method if other than guideline:

As in sub-chronic studies DBE appeared to affect rat olfactory tissue (female more than male), a female rat olfactory S-9 was used to eliminate the possibility that olfactory tissue may be uniquely metabolizing DBE to mutagenic metabolites.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium, other: TM677

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

0.0, 0.13, 0.21, 0.63, 1.07, 1.25, 2.13, 6.25 and 10.66 mmol/L in TA98 and TA100 assays with and without S9;
0, 0.28, 0.56, 2.82, 5.63 mM for TM677

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

additional positive controls substances: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

The negative control and positive indicators were assumed to be stable under the conditions of this study; no evidence of instability was observed.


S-9 ACTIVATION SYSTEMS

The 5-9 mix contained per mL: 0.3 mL of 5-9 diluted to 1.6 mg/mL with phosphate buffered saline (PBS) and 0.7 mL of a cofactor solution containing 8 micromoles MgCl2, 33 micromoles KCL, 5 micromoles glucose-6-phosphate, 4 micromoles NADP and 100 micromoles sodium phosphate
(pH 7.4). The rat liver S-9 (Sitek Research Laboratories. Rockville. MD Lot # 040187) was the 9,000 x g supernatant of liver homogenate (1 g wet liver: 3.0 mL PBS). The livers were obtained from 8 to 9 week old male Crl:CD@BR (Charles River, Kingston. NY) rats injected intraperitoneally
with Aroclor® 1254 (500 mg/kg) 5 days before sacrifice. The female rat olfactory S-9 was the 9.000 x g supernatant of olfactory homogenate (1 g wet olfactory tissue: 3 mL PBS). The olfactory tissue was obtained from 8 to 9 week old female Crl:CD@BR (Charles River, Kingston. NY) rats.
These rats were not pretreated with Arolcor.

REVERSE SUSPENSION ASSAY

The assay was performed in the presence and absence of a rat liver homogenate activation system (S-9 mix). Strains TA98 and TA100 were obtained from Dr. B. Ames. Berkeley. CA. Positive indicators and negative solvent controls were included in all assays. Overnight cultures of Salmonella typhimurium strains TA98 and TAI00 grown in Oxoid Nutrient Broth No 2 (NS) were diluted 1:4 with fresh NB. Treatments without activation(nonactivated) were conducted by adding
2.4 mL of diluted culture and 0.1 mL of the test sample in DMSO, control or positive indicator to 2.5 mL of NB. For activated treatments. 2.4 mL of diluted culture and 0.1 mL of the appropriate test sample was added to 2.5 mL S-9 mix (at a final protein concentration of 0.8 mg/5 mL). All
treatment tubes were glass and tightly sealed. The cultures were incubated for 2 hours in a 37°C shaking water bath (80-100 oscillations/min). The cultures were centrifuged at 3.000 rpm for 10 minutes and the cell pellet was resuspended in Davis Minimal Broth (DMB).

For mutagenesis evaluations. 1.0 ml of the cell suspension was added to 2 mL of molten top agar (0.6% Bacto-agar. 0.6% NaCl. 0.05 roM L-histidine. and 0.05 roM biotin). vortexed and poured on a Davis Minimal Base Agar (DMB containing 1.5% agar) plate. AlI exposures were plated in duplicate. The top agar was allowed to harden, plates inverted and incubated for approximately 36-48 hours at 37°C. Cytotoxicity experiments were run concomitantly by making dilutions (between 10^-4 and 10^-5) of the treatment suspensions using NB or phosphate buffered saline (PBS). Aliquots of 0.1 mL of the diluted cell sample were added to 2.0 mL of top agar, vortexed and poured on a Columbia Agar plate. The agar was allowed to harden. plates inverted and incubated for
approximately 18-36 hours at 37°C.

All colonies from individually labeled plates were counted with an Artek Automatic Colony counter. Mutant frequency was calculated by multiplying the average revertant count by the dilution factor for toxicity experiments then dividing by the average number of surviving cells for that dose group. In this report the frequency is expressed as the number of revertants per 10 cells. Only those trials meeting acceptability criteria for this assay are reported.


MICROFORWARD SUSPENSION ASSAY

Either a 0.1 mL aliquot-of quickly thawed or an overnight culture of TM677 was inoculated into 4.9 mL of DMB containing 0.05 mM biotin and 19 mg/mL dextrose. The culture was incubated at 37°C with shaking for 2 hours.
For cultures requiring activation 1 uL of test material and 10 uL of S-9 mix was added to 100 uL of bacterial culture. Test mixtures were incubated with gentle shaking at 37°C for 2 hours. PBS (0.4 mL) was then added to the reaction mixtures. An aliquot of 0.145 mL was removed and added to approximately 9.85 mL of 8 azaguanine (8-AG) supplemented top agar (0.6% purified agar. 100 mM NaCl, 120 mM sodium/potassium phosphate buffer pH 6.5. 0.05 mM biotin. and 0.5 mg/mL 8-AG). This was mixed and 2.5 mL was plated on purified agar (1.5% purified agar, 11 mM dextrose,
15 mM ammonium sulfate, 3 mM sodium citrate. 0.8 rmM magnesium sulfate. and 120 mM sodium/potassium phosphate buffer pH 6.5) plates in triplicate.

Plates were incubated for 48 hours at 37°C. Cytotoxicity was determined on the same samples. Aliquots of 0.145 mL were taken from the samples in the supplemented top agar and
added to approximately 4.85 mL PBS. Then 0.145 mL of this mixture was added to approximately 9.9 mL of top agar. This was vortexed and 2.5 mL was plated on Columbia agar plates in triplicate. All plates were allowed to harden, inverted and incubated for 24 hours at 37°C. Colonies from individually labeled plates were counted using an Artek
Automatic Colony Counter. Mutant frequencies were calculated by multiplying the average number of mutants in each dose group by the cytotoxicity dilution factor and then dividing by the number of surviving colonies. In this report mutant frequencies are expressed as mutants/l0 cells. Only those trials meeting acceptabililty criteria for this assay
are reported.

Evaluation criteria:

The guidelines below are used to aid in the evaluation and classification of a test sample along with sound scientific judgement and experience.
A test sample was classified as a POSITIVE when:
A) The number of induced revertants at one or more of the test sample concentrations studied are at least two times greater than the number of revertants in the solvent control. AND
B) There was a dose-response relationship.

A test sample is classified as a NEGATIVE when:
A) The criteria for a positive response is not met OR
B) There is no dose-response relationship.

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 examined

Positive controls validity:

valid

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 examined

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TM677

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

DBE exhibited no toxicity to strain TA98 or TA100 without and with rat liver activation up to a maximum dose of 8.53 mg DBE/treatment (10.7 mM or 1.7 mg/mL). No mutagenic responses were observed in either strain with or without activation in two independent trials. Mutant frequencies are shown in Tables 1 and 2.
Microforward Suspension Assay: DBE exhibited no toxicity to strain TM677 i n the presence of olfactory activation up to a maximum dose of 100 µg
DBE/treatment (5.63 mM or 0.9 mg/ml). No mutagenic responses were observed in two independent trials . Mutant frequencies are shown i n Table 3.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Mutagenic activity of DBE in S. thyphimurium strainTA98with/without metabolic activation (rat liver S9 mix)

Concentration [mmol/L]	without S9		with S9
	Trial 1^a)	Trial 2^a)	Trial 1^a)	Trial 2^a)
0.0 (control)	12.7	7.1	9.4	8.8
0.13		6.6		7.4
0.21	11.4		8.2
0.63		7.2		9.4
1.07	18.2	6.0	10.2
1.25				9.6
2.13	11.2		11.7
6.25		6.7		7.2
10.66	7.0		12.2
Positive control substances:
2-NF, 24 µM^b)	518	84
2-AA, 21 µM^b)			1052	1127

^a)Average value from two plates per concentration and trial

^b)2-NF: 2-Nitrofluorene, 2-AA: 2-Aminoanthracene

 

Table 2: Mutagenic activity of DBE in S. thyphimurium strain TM677 with metabolic activation (rat olfactory S9 mix)

Concentration [mmol/L]	without S9		with S9
	Trial 1^a)	Trial 2^a)	Trial 1^a)	Trial 2^a)
0.0 (control)	58.2	77.4	49.1	88.1
0.13		71.4		73.5
0.21	62.0		38.0
0.63		64.0		75.1
1.07	63.0		43.3
1.25		57.3		74.6
2.13	55.3		44.2
6.25		55.2		76.9
10.66	58.8		40.2
Positive control substances:
MNNG, 54 µM^b)	1441	1774
2-AA, 21 µM^b)			1347	1900

^a)Average value from two plates per concentration and trial

^b)MNNG: N-methyl-N'-nitro-N-nitrosoguanidine, 2-AA: 2-Aminoanthracene

 

Table 3: Mutagenic activity of DBE in S. thyphimurium strainTA100with/without metabolic activation (rat liver S9 mix)

Concentration [mmol/L]	with S9
	Trial 1^a)	Trial 2^a)
0.0 (control)	30	40
0.28	29	40
0.56	32
2.82	30	42
5.63	28	36
Positive control substances:
MNNG, 27 µM^b)	1096
2-AA, 9.3 µM^b)	300	348

^a)Average value from two plates per concentration and trial

^b)MNNG:N-methyl-N'-nitro-N-nitrosoguanidine, 2-AA: 2-Aminoanthracene

 

 

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

DBE is not mutagenic in the Ames reverse suspension assay with strains TA98 and TA100 and in the microforward suspension assay with strain TM677.

Executive summary:

The mutagenic potential of DBE has been assessed in the Salmonella thyphimurium/microsomal assay according to OECD guideline n° 471 and EC guideline n° B13/14, and in compliance with Good Laboratory Practice.

For the reverse suspension assay Salmonella thyphimurium strains TA98 and TA100 were used in presence and in absence of metabolic activation system from liver fraction of Aroclor 1254-induced rats (S9 mix). The test article was suspended in DMSO.

DBE was tested in 2 independent experiments performed according to the pre-incubation method in which bacteria were incubated with the test substance and S9 mix for 2 h at 37°C before platting.

Each strain of bacteria was exposed to dose-levels of 0.0, 0.13, 0.21, 0.63, 1.07, 1.25, 2.13, 6.25 and 10.66 mmol/L of the test item (two plates/dose-level). In each experiment, negative an positive controls were included using duplicate plates.

After 36 to 48 hours of incubation at 37°C, the number of revert ant colonies per plate was scored in each experiment, for each strain and for each experimental point. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or thinning of the bacterial lawn.

In the microforward susspension assay Salmonella thyphimurium strains TM677 was used in presence of

metabolic activation system from olfactory tissue homogenate fractions of uninduced female Crl:CD BR rats (S9 mix). The test article was suspended in DMSO. DBE was tested in 2 independent experiments performed according to the pre-incubation method in which bacteria were incubated with the test substance and S9 mix for 2 h at 37°C before platting (0, 0.28, 0.56, 2.82, 5.63 mM test substance concentration, three plates/dose-level). In each experiment, negative an positive controls were included using triplicate plates. After 24 hours of incubation at 37°C, the number of revert ant colonies per plate was scored in each experiment, for each strain and for each experimental point. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or thinning of the bacterial lawn.

No noteworthy toxicity was induced in any of the five tester strains.

Negative and positive controls responded adequately. The study was therefore considered valid.

The test substance DBE did not induce any noteworthy increase in the number of revertants which could considered as relevant, either with or without S9 mix, in any of the 3 tester strains.

The test item did not demonstrate any in vitro mutagenic activity in this bacterial test system, therefore DBE is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human, primary culture;

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 preparation

Test concentrations with justification for top dose:

Cytotoxicity assay: 0.00%, 0.01%, 0.1%, 0.3%, 0.6, and 1.0% (v/v), corresponding to 0.0, 0.11, 1.1, 3.3, 6.6, and 11.0 mg/mL, respectively.
Chromosome aberration assay: 0.00%, 0.04%, 0.10%, 0.3%, 0.4%, and 0.6 % (v/v), corresponding to 0.0, 0.44, 1.1, 3.3, 4.4, and 6.6 mg/mL, respectively;

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: in presence of S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: in absense of S9

Evaluation criteria:

Assessment of a test article as clastogenic is based on
(1) its ability to produce a statistically significant increase in chromosomal aberrations in test cultures as compared to the solvent control cultures at a minimum of one test level, and/or
(2) its ability to induce a significant dose-response. For increased statistical power, comparisons were made using the pooled data for untreated and solvent controls, provided they were not statistically different.

The complete chromosome aberration assay was based on the results of two independent, valid trials with activation and two without activation. An individual trial is considered valid if all of the following criteria are met:
The aberration frequencies of the negative control must fall in the range of historical control data.
The aberration frequencies for the positive indicator must be statistically significantly greater than the corresponding control frequency.
Analyzable preparations from at least 3 of the 4 test concentrations must be obtained in duplicate. Loss of an additional single replicate from any test point will not invalidate the trial. provided that both replicates at that test point are available for analysis from the other corresponding (activated / non-activated) trial.

Statistics:

Statistical significance was judged at the 5% level. For each trial, the proportion of abnormal cells and the proportion of cells with more than one aberration were evaluated using a Fisher Exact Test to compare each treatment level with the control, and a Cochran-Armitage test for linear trend (dose-response).

Species / strain:

lymphocytes: human; primary culture

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 0.6% (v/v) in culture medium

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: human, primary culture

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

no

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 0.6% (v/v) in culture medium

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY ASSESSMENT
Under non-activated conditons, DBE was cytotoxic at test concentrations above 0.3% (v/v) (Table 1). This was most evident in the reduced mitotic indices at 0.6% and 1.0% DBE relative to the DMSO controls. While the APT was not markedly increased at 0.6% DBE, there were fewer analyzable cells available. At 1.0% DBE, no analyzable cells were found. DBE was somewhat more cytotoxic under activated conditions; at 0.3%, the APT was increased by approximately 4-5 hr above control values, indicating significant cell cycle delay. No analyzable cells were obtained at higher DBE concentrations.

Based on these findings, the test concentrations selected for the non-activated chromosome aberration trials were 0.6, 0.4, 0.3 and 0.04% DBE (corresponding to concentrations of 6.6, 4.4, 3.3, and 0.44 mg/mL, respectively). For trials with activation, concentrations of 0.4, 0.3, 0.1 and 0.04% were chosen (corresponding to 4.4, 3.3, 1.1, and 0.44 mg/mL, respectively).

A harvest time of approximately 20 hr was judged appropriate for all cultures except the two highest activated DBE levels. As mitotic delay was anticipated, a later harvest time of approximately 24 hr was selected for these concentrations. Positive indicators were included at both sampling times.
 
CHROMOSOME ABERRATION STUDIES
Tables 2 and 3 give the frequency of structural aberrations per cell,  the percent abnormal cells and the percent cells with more than one aberration for the non-activated and activated trials, respectively.  Each data point represents the combined observations from two replicates (one male and one female donor). Notations of statistical significance in the tables refer to the Fisher Exact Test; comparisons are against the pooled untreated and solvent control data for a given trial.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Cytotoxicity Assessment

Proliferation kinetics of DBE-treated human lymphocytes

DBE conc. (v/v)	Donor	Cells scores	%MI	%MI+	% 2Mi	%M2+	%M3	APT (h)	Mitotic Index (%)
Nonactivated Treatment
0 (1% DMSO	M	50	44	16	40	0	0	17.2	4.0
	F	50	12	10	78	0	0	13.9	5.6
0.01%^a	M	-	-	-	-	-	-	-	-
	F	-	-	-	-	-	-	-	-
0.1%	M	-	-	-	-	-	-	-	-
	F	-	-	-	-	-	-	-	-
0.3%	M	50	34	8	58	0	0	15.7	7.0
	F	50	10	14	74	2	0	13.9	4.8
0.6%	M	50	58	10	32	0	0	18.6	1.8
	F	20^b	45	5	45	5	0	16.5	1.0
1.0%	M	Highly toxic; no analyzable cells							1.0
	F	Highly toxic; no analyzable cells							1.2
Activated Treatment
0 (1% DMSO	M	50	30	12	58	0	0	15.4	5.4
	F	50	30	14	54	2	0	15.4	3.8
0.01%	M	50	-	-	-	-	-	-	-
	F	50	-	-	-	-	-	-	-
0.1%	M	50	46	10	42	2	0	16.8	6.8
	F	50	32	12	56	0	0	15.8	6.0
0.3%	M	50	74	8	18	0	0	20.7	1.0
	F	50	62	18	20	0	0	19.5	4.0
0.6%	M	Highly toxic; no analyzable cells							1.0
	F	Highly toxic; no analyzable cells							1.0
	M	Highly toxic; no analyzable cells							1.0
	F	Highly toxic; no analyzable cells							1.0

A – Not analyzed since no cell cycle delay was observed at higher level.

B – Toxicity precluded scoring additional cells.

Table 2: Structural chromosome aberration in human lymphocytes in vitro

3-Hour DBE treatment without activation

DBE conc. (v/v)	Cells scored	Harvest time (h)	Aberrations / Cycle	% Abnormal Cells	% Cells with > 1 Aberr.	Mitotic Index (%)
Trial 1
0 (Untreated)	100	21	0.00	0.0	0.0	6.9
0 (1% DMSO)	100	21	0.05	2.0	1.0	5.3
0.04%	100	21	0.00	0.0	0.0	6.5
0.3%	100	21	0.20	3.0	2.0	3.2
0.4%	100	21	0.01	1.0	0.0	2.3
0.6%	100	21	0.01	1.0	0.0	2.6
MMC 0.25 ug/ml	100	21	0.26	20.0	3.0*	6.5
	100	25^a	0.23	16.0***	6.0**	6.8
Trial 2
0 (Untreated)	100	20	0.01	1.0	0.0	4.1
0 (1% DMSO)	100	20	0.02	2.0	0.0	3.1
0.04%	100	20	0.03	3.0	0.0	2.9
0.3%	100	20	0.00	0.0	0.0	2.6
0.4%	100	20	0.01	1.0	0.0	2.2
0.6%	100	20	0.01	1.0	0.0	1.6
MMC 0.25 ug/ml	100	20	0.04	4.0	0.0	3.6
	100	24^a	0.08	7.0	1.0	4.1

* p < 0.05; ** p < 0.01; *** p < 0.001;

a -  Data from later harvest of a concurrent MMC treatment provided to address the lack of positive responses with MMC in Trial 2 at 20 hours harvest time, where cell cycle delay may have been present.

Table 3: Structural chromosome aberrations in human lymphocytes in vitro.

3-Hours DBE treatment with activation

DBE conc. (v/v)	Cells scored	Harvest time (h)	Aberrations / Cycle	% Abnormal Cells	% Cells with > 1 Aberr.	Mitotic Index (%)
Trial 1
0 (Untreated)	100	21	0.01	1.0	0.0	7.6
0 (1% DMSO)	100	21	0.01	1.0	0.0	7.3
0.04%	100	21	0.02	2.0	0.0	7.6
0.1%	100	21	0.01	1.0	0.0	7,5
0.3%	100	25	0.14	5.0*	2.0	5.0
0.4%	71^a	25	0.27	11.3***	4.2*	0.2
CP 10 ug/mL	100	21	0.34	18.00***	9.0***	5.8
	100	25	0.93	37.00***	20.0***	3.3
Trial 2
0 (Untreated)	100	20	0.01	1.0	0.0	4.9
0 (1% DMSO)	50^b	20	0.02	1.0	0.0	4.0
0.04%	100	20	0.02	2.0	0.0	3.4
0.1%	100	20	0.01	1.0	0.0	3.7
0.3%	100	24	0.03	2.0	1.0	1.3
0.4%	61	24	0.15	11.5**	3.3	0.2
CP 10 ug/mL	100	20^c	0.07	5.0	2.0	3.5
	88	24	0.33	23.9***	5.7**	1.3

* p < 0.05; ** p < 0.01; *** p < 0.001;

A – Toxicity precluded scoring additional cells from male and/or female donor.

B – Test tube cracked during harvest; no cells available from male donor.

C – Lack of positive response was likely due to cell cycle delay at this sampling time.

Table 4: Structural chromosome aberratio in human lymphocytes in vitro. Confirmatory scoring of M1 cells from Cytotoxicity assessment. 3-Hour DBE treatment.

DBE conc. (v/v)	Cells scored	Harvest time (h)	Aberrations / Cycle	% Abnormal Cells	% Cells with > 1 Aberr.	Mitotic Index (%)
Nonctivated Treatment
0 (1% DMSO)	100	26	0.02	2.0	0.0	3.8
0.3%	100	26	0.02	2.0	0.0	4.6
0.6	42a	26	0.00	0.0	0.0	0.7
Activated Treatment
0 (1% DMSO)	100	25	0.05	3.0	2.0	5.3
0.3%	99a	25	0.40	15.2**	5.0	1.9

** p < 0.01

a – Toxicity precluded scoring additional cells from male and/or female donor.

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation
negative without metabolic activation

In the present in vitro chromosome aberrations test in primary human lymphocytes, under non-activated conditions, no statistically significant increases or concentration-related trends in chromosome aberrations were observed in either trial. Reduced mitotic indices at the higher DBE levels indicated that cytotoxic concentrations had been achieved.
 With S9 activation, statistically significant increases in the proportion of chromosomally-abnormal cells were seen at 0.3 and 0.4% DBE in Trial 1, and at 0.4% DBE in Trial 2. Additionally, the proportion of cells having more than one aberration was significantly increased at the 0.4% level in Trial 1. In both trials, statistically significant concentration-related trends were seen in the proportion of abnormal cells (p < 0.001, Trials 1 and 2) and the proportion of cells with more than one aberration (p < 0.01, Trial 1; p < 0.05, Trial 2. Mitotic indices were markedly reduced at the higher test concentrations.

Executive summary:

The mutagenic potential of DBE has been assessed in an in vitro Mammalian Chromosome Aberration Test according to OECD guideline n° 473 and EC guideline n° B10, and in compliance with Good Laboratory Practice.

Primary human lymphocytes were used in presence and in absence of metabolic activation system from liver fraction of Aroclor 1254-induced rats (S9 mix). The test article was suspended in DMSO. Cell were exposed to 0.00%, 0.04%, 0.10%, 0.3%, 0.4%, and 0.6 % (v/v), corresponding to 0.0, 0.44, 1.1, 3.3, 4.4, and 6.6 mg/mL, respectively (samples in duplicate: one male and one female donor). 44-47 hr after culture initiation the medium was replaced with treatment medium and the cultures were incubated for 3 hr at 37°C and rinsed with PBS. Fresh medium containing 5 -bromodeoxyuridine (BrdU) were then added and incubation continued for 24-26 hr, with Colcemid® (0.1 ug/mL) present during the final 2 hr to arrest cells in metaphase.Cells were then harvested, slides prepared, stained (Giemsa) and evaluated. An additional confirmatory scoring was carried out in a different laboratory. Rsults were statistically analysed.

Under nonactivated conditons, DBE was cytotoxic at test concentrations above 0.3% (v/v). DBE was somewhat more cytotoxic under activated conditions with significant cell cycle delay already at 0.3% (v/v).

Negative and positive controls responded adequately. The study was therefore considered valid.

Under non-activated conditions, no statistically significant increases or concentration-related trends in chromosome aberrations were observed in either trial. Reduced mitotic indices at the higher DBE levels indicated that cytotoxic concentrations had been achieved.

With S9 activation, statistically significant increases in the proportion of chromosomally-abnormal cells were seen at 0.3 and 0.4% DBE in Trial 1, and at 0.4% DBE in Trial 2. Additionally, the proportion of cells having more than one aberration was significantly increased at the 0.4% level in Trial 1. In both trials, statistically significant concentration-related trends were seen in the proportion of abnormal cells (p < 0.001, Trials 1 and 2) and the proportion of cells with more than one aberration (p < 0.01, Trial 1; p < 0.05, Trial 2. Mitotic indices were markedly reduced at the higher test concentrations.

DBE exhibited clastogenic activity in human lymphocytes under S9 activated conditions. In accordance with OECD Guideline 473 the test material is positive in the in vitro chromosome aberrations test in primary human lymphocytes.

A weight of evidence approach evaluating in vitro and in vivo data is therefore needed to assess the mutagenic potency of DBE.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The members of the category are all alcohol esters of dicarboxylic acids. All category members are manufactured by reacting an alcohol (methanol, butanol or isobutanol) with single dicarboxylic acids, succinic, glutaric or adipic acids or mixtures of these acids. The ester bonds are effectively metabolised by the body releasing the component alcohols and acids. The difference between members involves 3 parameters: 1) the alcohol used to esterify the acids, 2) the length of the acid molecule (4C, 5C or 6C) and 3) the presence of individual esters or mixtures thereof.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
The toxicity profile of the members (ecotoxicity and human health toxicity and the environmental fate) is consistent. All have low acute toxicity potential, are not sensitising, are mildly irritating to eyes and upper respiratory tract (where vapour pressure allows exposure), are not genotoxic or clastogenic (in vivo) and have minimal systemic toxicity. Data are available predominantly for the methyl esters (individual and mixture), dibutyl adipate and diisobutyl esters (mixture). Within the category, read across is used to cover the higher tier human health toxicity studies predominantly.

See attached document with the justification for the category/read-across approach.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The test methods used in this study were mutagenicity systems regarding reverse mutation from histidine auxotrophy to histidine prototrophy in Salmonella typhimurium and reverse mutation from tryptophan auxotrophy to tryptophan prototrophy in Escherichia coli as indices.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: maintained since October 31, 1975
- Properly maintained: yes
- These bacterial strains were stored frozen at −80°C or below. At preparation of frozen bacterial suspensions, characterization of the strains was conducted for the amino acid auxotrophy, UV sensitivity, membrane mutation (rfa) and ampicillin resistance factor (pKM101).
- Seed bacteria were inoculated into L-shaped test tubes containing Nutrient Broth No. 2 (Oxoid Ltd.) and incubated by reciprocal shaking at 37°C for approximately 10 hours to prepare bacterial suspensions.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: maintained since October 31, 1975
- Properly maintained: yes
- These bacterial strains were stored frozen at −80°C or below. At preparation of frozen bacterial suspensions, characterization of the strains was conducted for the amino acid auxotrophy, UV sensitivity, membrane mutation (rfa) and ampicillin resistance factor (pKM101).
- Seed bacteria were inoculated into L-shaped test tubes containing Nutrient Broth No. 2 (Oxoid Ltd.) and incubated by reciprocal shaking at 37°C for approximately 10 hours to prepare bacterial suspensions.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose range finding study - 50 - 5000 μg/plate
Main study - 312.5 - 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: As per guidleines, DMSO is a recommended vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Remarks:

AF2: Furylfuramide (Lot No. 46, purity 99.9%), SA: Sodium azide (Lot No. TWR3330, purity ≥90%), 9AA: 9-Aminoacridine (Lot No. 96F05641, purity ≥98%), 2AA: 2-Aminoanthracene (Lot No. DSF2950, purity ≥90%)

Positive control substance:

other: AF2: Furylfuramide (Ueno Fine Chemicals Industry, Ltd.), SA: Sodium azide (Wako Pure Chemical Industries, Ltd.) , 9AA: 9-aminoacridine (Sigma Chem. Co.), 2AA: 2-aminoanthracene (Wako Pure Chemical Industries, Ltd.)

Remarks:

AF2 and 2AA were dissolved in DMSO (Wako Pure Chemical Industries, Ltd.) and stored frozen at −20°C. These solutions were thawed at each use. 9AA and SA were dissolved in DMSO and distilled water, respectively, and promptly used for the study

Details on test system and experimental conditions:

Reverse mutation assays were conducted by the plate method for the direct and metabolic activation methods. Top agar (2 ml), each test substance formulation (0.1 ml), phosphate buffer (0.5 ml; 0.5 ml of S9 mix for the metabolic activation method) and each bacterial suspension (0.1 ml) were mixed in small test tubes. The mixtures were poured and solidified on the synthetic agar plate medium. For the control groups, DMSO or several positive control solutions were used instead of the test substance formulations. The agar plates were incubated at 37°C for 48 hours and the number of revertant colonies was calculated. The presence/absence of the antibacterial effects of DBA was judged based on the conditions of the bacterial film on the surface of the agar plates macroscopically or under a stereoscopic microscope. Three plates/group for the negative and positive control groups and 1 plate/dose for the test substance groups were used in the dose range-finding test. In the main tests, 3 plates/group for the negative and positive control groups and 3 plates/dose for the test substance groups were used, and the mean values and standard deviations were calculated for each group. The dose range-finding test was conducted once, while the main tests were conducted twice at the same dose levels to confirm the reproducibility of the results.

Evaluation criteria:

The test substance was judged to possess the mutagenic potential (positive) in the test system used in this study when the mean numbers of revertant colonies in the test substance plates were greater than twice the negative control values and reproducibility or dose-dependency was evident in the increases, in at least 1 of the 5 bacterial strains in either the direct or metabolic activation method.

Statistics:

Standard statistical methods were employed.

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:

valid

True negative controls validity:

not examined

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:

valid

True negative controls validity:

not examined

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only at highest dose level tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

valid

Positive controls validity:

valid

Additional information on results:

none

Dose range-finding test - When the dose range-finding test was conducted at the dose levels ranging from 50 to 5000 μg/plate using a common ratio of approximately 3, no antibacterial effects of dibutyl adipate were observed at any dose level in any bacterial strain. Therefore, the highest dose level for the main tests was selected at 5000 μg/plate for both the direct and metabolic activation methods in all bacterial strains. 

Main tests - The main tests were conducted at the dose levels ranging from 312.5 to 5000 μg/plate using a common ratio of 2 for both the direct and metabolic activation methods in allbacterial strains. Dose-dependent increases in the number of revertant colonies were not noted in any of the 5  bacterial strains in either the direct or metabolic activation method in either main test. Antibacterial effects of dibutyl adipate were observed only at 5000 μg/plate for TA100 in the direct method. 

 

In the bacterial reverse mutation test of Dibutyl Adipate, increases in the number of revertant colonies were evident in all bacterial strains in the positive control groups, and the numbers of revertant colonies in the negative and positive control groups were within the ranges of historical control data. Therefore, the validity of the test system used in this study was confirmed

Conclusions:

Interpretation of results: negative. Based on these results, dibutyl adipate (DBA) was judged not to possess the mutagenic potential (negative) in the test system used in this study.

Executive summary:

A bacterial reverse mutation test of dibutyl adipate was conducted to investigate its mutagenic potential.  When a dose range-finding test was conducted at dose levels of 50-5000 μg/plate using bacterial strains of Salmonella typhimurium (TA100, TA1535, TA98 and TA1537) and Escherichia coli (WP2 uvrA), no antibacterial effects were observed by the direct or metabolic activation method. Therefore, the main tests were conducted at dose levels of 312.5-5000 μg/plate.  Increases in the number of revertant colonies of greater than twice the negative control values were not noted at any dose level in any of the 5 bacterial strains in either main test. Therefore, dibutyl adipate was judged not to possess the mutagenic potential (negative) in the test system used in this study.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The members of the category are all alcohol esters of dicarboxylic acids. All category members are manufactured by reacting an alcohol (methanol, butanol or isobutanol) with single dicarboxylic acids, succinic, glutaric or adipic acids or mixtures of these acids. The ester bonds are effectively metabolised by the body releasing the component alcohols and acids. The difference between members involves 3 parameters: 1) the alcohol used to esterify the acids, 2) the length of the acid molecule (4C, 5C or 6C) and 3) the presence of individual esters or mixtures thereof.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
The toxicity profile of the members (ecotoxicity and human health toxicity and the environmental fate) is consistent. All have low acute toxicity potential, are not sensitising, are mildly irritating to eyes and upper respiratory tract (where vapour pressure allows exposure), are not genotoxic or clastogenic (in vivo) and have minimal systemic toxicity. Data are available predominantly for the methyl esters (individual and mixture), dibutyl adipate and diisobutyl esters (mixture). Within the category, read across is used to cover the higher tier human health toxicity studies predominantly.

See attached document with the justification for the category/read-across approach.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

This in vitro chromosomal aberration test was conducted using cultured Chinese hamster (CHL/IU) cells to evaluate the potential cytogenetic effects of dibutyl adipate on cultured cells.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Frozen CHL/IU cells derived from Chinese hamsters and obtained from Japanese Collection of Research Bioresources (JCRB) in February 1988 (passage numbers were 4 at receipt and 12 at present; hereafter referred to as “CHL cells”) were thawed and used for the study within the passage number of 10.
CHL cells (2 × 104 cells) were seeded in culture dishes (diameter 6 cm, Corning Incorporated) containing 5 ml of culture medium and incubated in a CO2 incubator (5% CO2) at 37°C.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The concentrations of the test substance formulations were 0.08-2.60 mg/ml (10 mM) in the direct method and the metabolic activation method with the S9 mix, while 0.006-0.200 mg/ml in the metabolic activation method without the S9 mix since marked growth inhibition was noted at a concentration range of 0.160 mg/ml or above in a preliminary study. Two dishes were used for each concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: As per guidelines, DMSO is a recommended vehicle

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Mitomycin C and cyclophosphamide

Details on test system and experimental conditions:

Preparation of culture medium - Eagle’s MEM liquid medium containing fetal calf serum (FCS, Lot No. 1C2073, JRH Biosciences) at 10% was used for culturing. The MEM liquid medium was prepared as follows: 9.4 g of Eagle’s MEM medium powder “Nissui” (1) (Nissui Pharmaceutical Co., Ltd.) were dissolved in 1 liter of distilled water; this solution was autoclaved at 121°C for 15 minutes; and 300 mg of sterile L-glutamine (Nissui Pharmaceutical Co., Ltd.) and approximately 12.5 ml of a 10% NaHCO3 solution were added to the solution. The MEM liquid medium at 2-fold concentration was prepared by dissolving 9.4 g of the above medium in 500 ml of distilled water and the subsequent procedures were carried out in a similar manner.

Culture conditions - CHL cells (2 × 104 cells) were seeded in culture dishes (diameter 6 cm, Corning Incorporated) containing 5 ml of culture medium and incubated in a CO2 incubator (5% CO2) at 37°C.

Preparation of the test substance formulations - The test substance formulations were prepared fresh at each use. DMSO (Lot No. 12H0658, Sigma Chemical Co.) was used as the solvent. The bulk substance of the test substance was dissolved in the solvent to yield stock solutions (40 and 520 mg/ml for the cell growth inhibition test and 9.2 and 520 mg/ml for the chromosomal aberration test). The stock solutions were serially diluted with the solvent to yield the specified concentrations of test substance formulations. The test substance formulations were added to the culture medium at a fixed concentration of 0.5% (v/v) in all assays. In the chromosomal aberration test, the test substance formulations were analyzed for the content in the low and high concentration groups in the direct and metabolic activation methods by the Laboratory of Analytical Chemistry, Hatano Research Institute. All concentrations of the test substance formulations were within the acceptable range (mean content in the solvent was 90.0-110% of the volume added to the medium.

Test conditions - In the direct method, the cells were incubated for 3 days and the culture medium was discarded. Fresh medium (5 ml) and each test substance formulation (25 μl) were added to the dishes and the cells were further incubated continuously for an additional 24 and 48 hours.
In the metabolic activation method, the cells were incubated for 3 days and the culture medium was discarded. Three ml of a solution which was prepared by mixing the MEM medium, 2-fold concentration of MEM medium and S9 mix at a ratio of 4/1/1 was added to the dishes. For the groups treated without the S9 mix, only 3 ml of the MEM medium were added to the dishes. Then, 15 μl of each test substance formulation were added and the cells were incubated for an additional 6 hours. After completion of incubation, the medium was changed with fresh one and the cells were further incubated for 18 hours.

Treatment conditions - A cell growth inhibition test was conducted for the groups in the 48-hour treatment by the direct method and the groups by the metabolic activation method with and without the S9 mix. The concentrations of the test substance formulations were 0.08-2.60 mg/ml (10 mM) in the direct method and the metabolic activation method with the S9 mix, while 0.006-0.200 mg/ml in the metabolic activation method without the S9 mix since marked growth inhibition was noted at a concentration range of 0.160 mg/ml or above in a preliminary study. Two dishes were used for each concentration.
Method of specimen preparation - After completion of incubation, the culture medium was discarded and 10% formalin was added to fix the cells adhered to the dishes in situ. After fixation, the cells were stained with 0.1% crystal violet.

Preparation method of chromosomal specimens - At 2 hours prior to completion of the incubation, colcemid was added to the culture medium at a final concentration of approximately 0.1 µg/ml. After the full incubation period, the cells in each group were rinsed with Ca2+- and Mg2+-free phosphate buffer, detached from the dishes by pipetting and collected into 10 ml centrifuge tubes. The tubes were centrifuged at 1000-1200 rpm for 5 minutes and the supernatant fractions were discarded. Three ml of a 0.075 M KCl aqueous solution were added to precipitated cells to treat the cells hypotonically for approximately 30 minutes. After the hypotonic treatment, approximately 6 ml of Carnoy’s fluid (glacial acetic acid/methanol = 1/3 v/v) were added to the top portion of the hypotonic solution and mixed by gently pipetting from its bottom portion to fix the cells. The tubes were then centrifuged at 1000-1200 rpm for 5 minutes. The supernatant fractions were removed, fresh Carnoy’s fluid was added again to re-suspend the cells by pipetting and the tubes were further centrifuged at 1000-1200 rpm for 5 minutes. This procedure was repeated several times. Carnoy’s fluid (0.2-0.5 ml) was added to white cell pellets obtained by centrifugation to suspend the cells thoroughly. Small quantities of the cell suspensions were pipetted onto previously washed glass slides and air-dried. Six slides were prepared from each dish. The ID No. of the test system, Code No. and Slide No. were recorded on the frosted portion of each slide with pencils. The dried slides were stained with a stain solution, which was prepared by diluting 4.5 ml of Giemsa stock solution (Merck) with 150 ml of M/15 phosphate buffer (pH 6.8), for approximately 8 minutes, lightly rinsed with distilled water and air-dried. The stained slides were placed in slide cases in order of the Code Nos. The cases were clearly denoted with the ID No. of the test system and the date of slide preparation and stored.

Evaluation criteria:

Of the prepared slides, several slides obtained from the same dishes were analyzed by multiple observers under coded conditions not to find out the treatment conditions by the observers. Metaphase cells with well-spread but not so scattered chromosomes were selected and the position of cells with aberrations on the slides was recorded in record forms as the position on the microscope stage.
Chromosomal analysis was conducted based on the classification method established by the Japanese Environmental Mutagen Society, Mammalian Mutagenicity Study (MMS) Group1). The cells were examined for structural aberrations, such as chromosome/chromatid gaps, breaks, exchanges, etc., and for polyploidy. For structural aberrations and polyploidy, 200 and 800 metaphase cells per group were examined, respectively

Statistics:

Analytical results in the untreated, solvent and positive control groups and test substance treated groups were tallied up for the number of cells analyzed, categories and the number of structural aberrations and the number of polyploidy, and values in each group were recorded on the record forms.
The incidences of cells with chromosomal aberrations were analyzed for statistically significant differences using Fisher’s exact probability test between the solvent control and test substance treated groups or the positive control groups.
Final judgment of the potential of the test substance to induce chromosomal aberrations was made according to the criteria by Ishidate, et al. When the incidences of cells with chromosomal aberrations were <5%, ≥5% but <10%, and ≥10%, the results were judged to be negative, false positive and positive, respectively. If false positive results were obtained, the reproducibility, dose-dependency, etc. were determined by a chromosomal aberration test or a micronucleus test prior to the final judgment

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

None

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation The effects of dibutyl adipate to induce chromosomal structural aberrations were evident at the low and mid concentrations by the metabolic activation method with the S9 mix and the results were judged to be positive

DBA did not show the potential to induce chromosomal structural aberrations or polyploidy in any of the groups, in which the cells were continuously treated for 24 hours (0.7-2.6 mg/ml) or 48 hours (0.7-2.6 mg/ml) by the direct method, including the groups at a concentration of 10 mM.
Conversely, in the metabolic activation method, chromosomal structural aberrations (including gaps) were evident in 6.5% and 12.0% of the cells analyzed at the low (0.7 mg/ml) and mid (1.3 mg/ml) concentrations with the S9 mix.
Therefore, it was concluded that DBA was activated by metabolization and induced chromosomal aberrations in in vitro CHL cells under the above experimental conditions

Executive summary:

The potential of dibutyl adipate to inducein vitro chromosomal aberrations was evaluated using cultured Chinese hamster (CHL/IU) cells and positive results were obtained. In the direct method and the metabolic activation method with S9 mix, cell-growth inhibition obviously exceeding 50% was not observed even at a concentration of 10 mM (2.6 mg/ml). Meanwhile, in the metabolic activation method without the S9 mix, the concentration obviously exceeding the 50% cell-growth inhibitory concentration (approx. 60% inhibitory concentration) was 0.046 mg/ml. Therefore, the maximum concentrations were selected at 2.6 mg/ml for the direct method and the metabolic activation method with the S9 mix and 0.046 mg/ml for the metabolic activation method without the S9 mix. One half and one quarter of the maximum concentrations were selected as the mid and low concentrations, respectively. After 24- and 48-hour continuous treatment by the direct method without the S9 mix and 6-hour treatment (plus an 18-hour recovery period) by the metabolic activation method with and without the S9 mix, chromosomal slides were prepared and examined by microscopy to evaluate the potential of dibutyl adipate to induce chromosomal aberrations. No effects of dibutyl adipate to induce chromosomal structural aberrations or polyploidy were evident in any group in which CHL/IU cells were treated for 24 or 48 hours by the direct method or for 6 hours by the metabolic activation method without the S9 mix. Conversely, the effects of dibutyl adipate to induce chromosomal structural aberrations were evident at the low and mid concentrations by the metabolic activation method with the S9 mix and the results were judged to be positive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

As a result of the positive assay in the presence of metabolic activation,in vivo studies have been performed on dimethyl glutarate and the mix of methyl esters. Both of these assays demonstrate a clear absence of clastogenicity. The in vivo assays are considered to be more representative and more predictive of the potential human situation. Therefore, the weight of evidence for these compounds is that they are not clastogenic in vivo. As such it is concluded that the mix of dibutyl esters will also be negative for clastogenicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The members of the category are all alcohol esters of dicarboxylic acids. All category members are manufactured by reacting an alcohol (methanol, butanol or isobutanol) with single dicarboxylic acids, succinic, glutaric or adipic acids or mixtures of these acids. The ester bonds are effectively metabolised by the body releasing the component alcohols and acids. The difference between members involves 3 parameters: 1) the alcohol used to esterify the acids, 2) the length of the acid molecule (4C, 5C or 6C) and 3) the presence of individual esters or mixtures thereof.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
The toxicity profile of the members (ecotoxicity and human health toxicity and the environmental fate) is consistent. All have low acute toxicity potential, are not sensitising, are mildly irritating to eyes and upper respiratory tract (where vapour pressure allows exposure), are not genotoxic or clastogenic (in vivo) and have minimal systemic toxicity. Data are available predominantly for the methyl esters (individual and mixture), dibutyl adipate and diisobutyl esters (mixture). Within the category, read across is used to cover the higher tier human health toxicity studies predominantly.

See attached document with the justification for the category/read-across approach.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Crl:DC-1 (ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Cr1:Cne-l(ICR)BR mice (43 days old) were from Charles River, Kingston, NY. They were quarantined and acclimated to laboratory conditions for at least 6 days prior to initiation of the study. Except during exposure, the animals were housed in standard 4" x 7'0 x 5" wire mesh cages, with Purina Certified Rodent and tap water supplied ad libidum. Animal rooms were maintained on a timer controlled 12-hr-light/dark cycle. Environmental conditions of the rooms were targeted for a temperature of 23 + 2°C and relative humidity of 50 + 10%. Any excursions outside these ranges were of small magnitude and/or brief duration and did not adversely affect the validity of the study. During the quarantine period, the mice were housed three per cage; during testing, they were housed individually. All animals were uniquely identified by individual cage numbers and colored markings on their tails

Route of administration:

inhalation: aerosol

Vehicle:

- Physical State: liquid
- Vehicle(s)/solvent(s) used: air;
- Justification for choice of solvent/vehicle: used for aerosol generation
- Concentration of test material in vehicle: 5.5, 11.0, and 19.0 mg/L DBE in aerosol air

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

In the rangefinding studies aerosol atmospheres of DBE were generated with either a Spraying Systems or Solosphere8 nebulizer.

All atmospheres were generated for the micronucleus assay using the Solosphere8 nebulizer. With the Spraying Systems nebulizer, liquid DBE was pumped into the nebulizer with a Harvard Model 975 compact infusion pump. Air introduced at the nebulizer aerosolized the liquid DBE. and swept the aerosol stream into the exposure chamber. With the Solophere8 nebulizer. liquid DBE was drawn from a reservoir by capillary action into a high pressure air stream and was aerosolized off a spherical glass bead. For both methods, the resulting aerosol stream was dispersed with a baffle as it entered the chamber.

The chamber exhausts were drawn through scrubbers containing acetone, dry-ice cold traps and MSA cartridge filters prior to being discharged into the exhaust hoods. For the control chamber, air only was pumped into a similar exposure chamber.

Atmosphere Analyses: The atmospheric concentration of DBE in each exposure chamber was monitored at approximately 30 minute intervals. Known volumes of the chamber atmosphere were drawn from the animals' breathing zones through 2 fritted glass midget impingers containing acetone as a with a Hewlett Packard 5710A gas chromatograph equipped with a flame ion trapping solvent. The resulting solutions were analyzed in duplicate. The injection port and detector temperatures were 250°C. Nitrogen was used as the carrier gas. Samples were chromatographed isothermally at 125°C on a 3 ft. x 2 mm 10 glass column packed with 10% SP-I000 on 100/120 mesh Chromosorb W-AW.

Atmospheric concentrations of DBE were determined by comparing the detector responses against standard curves calculated by regression. Standards were prepared as needed by diluting known amounts of liquid DBE in acetone. Particle size (mass median aerodynamic diameter and percent respirable) was determined during each exposure with a Sierra Series 210 Cascade Impactor. During each exposure, chamber temperatures were monitored with mercury thermometers. Relative humidities were measured with a Vaisala Model HMI 31F Temperature and Humidity Indicator. Chamber oxygen concentrations were determined with a Biosystems Model 3100R oxygen monitor.

Duration of treatment / exposure:

One day prior to exposure, mice (approximately 7 weeks old) were assigned to exposure groups with equivalent mean body weights (sexes separate) using a computer-based algorithm. The mean pre-treatment weight of the males was 34 g (range 32-38 g) and the females was 25 g (range 20-28 g). Two groups of 15 male and 15 female mice (low and intermediate dose groups) and one group of 18 male and 18 female mice (high dose group) were restrained and exposed as described for the range-finding study to concentrations of DBE for 6 hrs. One control group of 15 male and 15 female mice were similarly exposed to air only for 6 hrs. Concurrent with the initiation of these exposures, 5 male and 5 female mice were dosed by intraperitoneal injection with 20 mg/kg CP, the positive indicator. The mice were observed for clinical signs of toxicity during and immediately following exposure. Surviving mice were weighed and observed daily until sacrifice. Approximately 24, 48 and 72 hrs after initiation of exposure. mice were sacrificed by CO2 asphyxiation.

Frequency of treatment:

1 time, 6 hours;

Post exposure period:

10 to 13 days;

Remarks:

Doses / Concentrations:
range-finding study: 6.9, 11.0, 13.0, and 19 mg/L in aerosol; in micrunucleus test: 5.0, 10.0, and 23 mg/L in aerosol;
Basis:
nominal conc.

No. of animals per sex per dose:

In range finding: 15 males, 15 females per group;
In micronucleus test: 5 males, 5 females per group:

Control animals:

yes

Positive control(s):

cyclophosphamide;
- Route of administration: intraperitoneal (i.p.);
- Doses / concentrations:Stock solution: 2.9 mg/L in 0.9% sterile saline; effective dose applied = 20 mg/kg.

Details of tissue and slide preparation:

Immediately following sacrifice, the marrow from both femurs of each animal was aspirated and flushed into 5 mL prewarmed (37°C) fetal bovine serum (Hyc1one Laboratories, Lot 1111536 or Gibco Laboratories, Lot 56K6455). The marrow button was collected by centrifugation (IEC Centrifuge Model HNS-II) at approximately 1000 rpm for 5 min. Most of the supernatant was removed and the cells were resuspended in the remaining 1-2 drops of serum. A Miniprep. Automatic Blood Smearing Instrument was used to make the bone marrow smears. Three slides per animal were prepared and fixed in absolute methanol for 5 min. The slides were stained for 2.5 min in acridine orange (Sigma Chemical Co., Lot 73F-3673) at a concentration of 0.042 mg/mL in phosphate buffer (pH 7.4), followed by phosphate buffer rinses. Immediately prior to scoring, a coverslip was floated on each slide using phosphate buffer.

Evaluation criteria:

Representative slides from each animal were examined in a blind manner using incident light fluorescence microscopy (Leitz Dialux 22 microscope with Ploempak and H-2 filter cube). Only cells showing good morphology and staining were selected for scoring. PCEs were identified by their characteristic reddish staining; NCEs appeared dark green. One thousand PCEs per animal were scored for the presence of micronuclei, which are typically round, bright yellow-green staining bodies.
Inclusions in PCEs which were irregularly shaped or stained, or were not
in the focal plane of the cell were judged to be artifacts and were not
scored. Cells containing more than one micronucleus were counted as
having a single micronucleus; the unit of scoring was the micronucleated
PCE, not the micronucleus. The number of micronucleated NCEs seen in the
optic fields scored to obtain 1000 PCEs was also recorded. The ratio of
PCEs to NCEs was determined for each animal based on 1000 total
erythrocytes.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

During exposure, no adverse clinical signs were observed in the 5.5 mg/L-treated mice. Mice exposed to 11 mg/L had no response to sound. At 19 mg/L, observations could not be made due to the dense aerosol cloud. When released from restrainers after exposure, mice in all groups (including controls) were wet and stained by feces. One control mouse had bloody hindquarters, presumably caused by struggling in the restrainer. In addition, 5 male mice (1 low dose and 4 high dose) and 2 female high dose mice were found dead upon unloading the animals from the exposure chambers. These deaths were believed to be restrainer-related.

At 24 hrs post-exposure, 25 of the surviving animals had ruffled fur, with the greatest number of animals (10 males and 4 females) exhibiting this condition in the high DBE exposure group. Additionally, the following clinical signs were observed at 24 hrs post-exposure: labored breathing in 3 high dose males, tremors, slow movement, and diarrhea each in 1 high dose male, yellow stained fur in 1 low dose male and hypersensitivity in 1 low dose female. Ruffled fur was present among fewer animals at the 48- and 72-hr observation periods; the only additional clinical sign exhibited was
hunched posture by a high dose male.

 

When compared to the concurrent negative controls, significant weight loss was seen in all of the DBE exposed males at the 24-hr sacrifice time, the high dose 48- and 72-hr males, the high dose 24-hr females and the low dose 72-hr females. No significant weight loss or decreased weight gain was observed in any other test group.

 

Table 2 summarizes the data for micronucleated PCEs and PCE:NCE ratios. Individual animal data for the 24, 48, and 72-hr sacrifice groups are shown in Appendices A, B, and C, respectively. Significant depression of the PCE:NCE ratios was seen in the 24-hr 11 mg/L- and 19 mg/L-treated females. There were no statistically significant differences in the proportion of micronucleated PCEs between DBE-treated mice and the concurrent negative control groups at any sampling interval, and no statistically significant concentration-related trends were present. In comparison, both the CP-treated males and females showed significant increases in micronucleated PCEs compared to the concurrent air controls.

Table 2: Micronucleaus Data Summary

DBE (mg/L	Mean MN-PCEs +/- S.E. (1000 PCEs Scored)							Mean Ratio PCEs / NCEs +/- S.E.
Males
	Sacrifice time							Sacrifice time
	N	24h	N	48h	N	72h		24h	48h	72h
0	5	1.6 ± 0.2	5	1.6 ± 0.6	5	1.6 ± 0.6		0.9 ± 0.2	0.8 ± 0.2	0.9 ± 0.1
5.5	5	1.8 ± 0.8	4	1.0 ± 0.4	5	1.8 ± 0.7		0.8 ± 0.1	0.7 ± 0.1	0.8 ± 0.1
11.0	5	2.0 ± 0.3	5	1.4 ± 0.9	5	1.4 ± 0.5		0.6 ± 0.1	0.8 ± 0.1	0.7 ± 0.1
19	5a	3.0 ±-0.8	5a	1.8 ± 0.6	4b	1.5 ± 0.3		0.7 ± 0.1	0.8 ± 0.1	0.5 ± 0.1
Trend	N.S.		N.S.		N.S.
CP	9.6 ±1.5***							0.8 ±0.1
Females
0	5	1.4 ± 0.4	5	3.2 ± 0.7	5	1.6 ± 0.8		1.2 ± 0.1	0.8 ± 0.2	1.0 ± 0.2
5.5	5	1.8 ± 0.4	5	1.4 ± 0.5	5	2.2 ± 1.5		0.9 ± 0.1	0.9 ± 0.1	0.7 ± 0.1
11.0	5	1.0 ± 0.5	5	1.0 ± 0.4	5	1.6 ± 0.5		0.7 ± 0.1**	0.9 ± 0.2	0.6 ± 0.0
19.0	5a	1.6 ± 0.7	6	2.3 ± 0.7	5a	1.8 ± 0.6		0.7 ±0.0*	0.8 ± 0.1	.07 ± 0.1
Trend	N.S.		N.S.		N.S.
CP	15.4 ± 2.0***							1.0 ± 0.1

MN-PCEs, micronucleated polychromatic erythrocytes; NCEs, normochromatic erythrocyte; CP,  cyclophospamide, 20 mg/kg, i.p.

a, one mouse found dead before scheduled sacrifice time;

a, two mice fond dead before scheduled sacrifice time.

*, p 005; **,  p 0.01; ***, p 0.001; N.S., not significant;

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of this assay, DBE did not induce micronuclei in bone marrow cells of mice; the compound was negative in this in vivo test.

Executive summary:

A mouse bone marrow micronucleus test similar to the OECD Guideline 474 was performed under GLP regulation. While significant depression of the micronucleated polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) ratios was seen in the 24-hr 11 mg/L- and 19 mg/L DBE-treated females, there were no statistically significant differences in the proportion of micronucleated PCEs between DBE-treated mice and the concurrent negative control groups at any sampling interval, and no statistically significant concentration-related trends were present. In comparison, both the cyclophosphamide (CP)-treated males and females showed significant increases in micronucleated PCEs compared to the concurrent air controls. Under the conditions of this assay, DBE did not induce micronuclei in bone marrow of mice and the compound ws classified negative in this in vivo test.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The members of the category are all alcohol esters of dicarboxylic acids. All category members are manufactured by reacting an alcohol (methanol, butanol or isobutanol) with single dicarboxylic acids, succinic, glutaric or adipic acids or mixtures of these acids. The ester bonds are effectively metabolised by the body releasing the component alcohols and acids. The difference between members involves 3 parameters: 1) the alcohol used to esterify the acids, 2) the length of the acid molecule (4C, 5C or 6C) and 3) the presence of individual esters or mixtures thereof.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
The toxicity profile of the members (ecotoxicity and human health toxicity and the environmental fate) is consistent. All have low acute toxicity potential, are not sensitising, are mildly irritating to eyes and upper respiratory tract (where vapour pressure allows exposure), are not genotoxic or clastogenic (in vivo) and have minimal systemic toxicity. Data are available predominantly for the methyl esters (individual and mixture), dibutyl adipate and diisobutyl esters (mixture). Within the category, read across is used to cover the higher tier human health toxicity studies predominantly.

See attached document with the justification for the category/read-across approach.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female animals weighed between 152.1-173.6 grams and 125.7 and 148.6 grams, respectively. Animals were randomly assigned to groups with sexes separated. Groups, n=5/sex/exposure group, were kept in cages with a controlled environment. The temperature was kept at 21°C with a relative humidity of 50%. The room was illumiated for 12 hours per day. Animals were provided food and tap water ad libitum except when it was witheld prior to exposure. Aniamls were acclimatized to at least 12 days prior to study initiation.

Route of administration:

inhalation: aerosol

Vehicle:

None

Details on exposure:

Liquid test material was delivered to a concentric jet atomiser and generated as droplets into a stream of dry air for administraion to the rats in whole body exposure chambers. Exposure concentration was achieved by meteringthe test substance from polypropylene syringes mounted on syringe drivers.

Exposures were:
0 mg/L (Air) - Negative control
0.5 mg/L (0.545 analytical)
1.0 mg/L (0.807 anayltical)
2.0 mg/L (2.298 analytical)
20 mg/kg Cyclophosphamide (positive control)

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

2 days

Post exposure period:

24 hours

Remarks:

Doses / Concentrations:

Basis:
analytical conc.

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes

Positive control(s):

Cyclophosphamide 20 mg/kg adminstered by oral gavage at the end of the second exposure treatment

Tissues and cell types examined:

Femoral bone marrow

Details of tissue and slide preparation:

Following sacrifice of animals, Both femurs of each animal were removed and bone marrow extracted and pooled into 10 mL of salt solution. The cell suspensions were centrifuged at 1000g for 5 minutes. The pellet was resuspended in 2 ml of filtered fetal calf serum to facilitate smearing on glass slides. Several smears were prepared from each femur. A modified Feulgen staining method was used which stains DNA-containing bodies deep purple without staining mast cell granules. The method also allows for differentiation of mature and immature erythrocytes.

Evaluation criteria:

Slides were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes. Micronuclei were identified with the following criteria:
- large enough to discern morphological characteristics
-should be generally rounded in shape with clearly defined outline
-should be deeply stained and in color similar to other nuclei
-should lie in the same focal plane as the cell
-lack internal structure
-there should be no micronulceus like debris surrounding the cell

A portion of immature erythrocytes for each animal was asessed by examination of at least 1000 erythrocytes.

A positive repsonse is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared to controls (p<0.01) and exceed laboratory historical control ranges.

Bone marrow cell toxicity is indicated by a substantial and statistically significant dose-related decrease in the proportion of immature erythrocytes (p < 0.01).

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

lethargy, piloerection, partially closed eyes, brown staining

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Treatment	Exposure level	Percent Immature Erythrocytes	Number of micronucleated cells per 2000 Immature Erythrocytes	Number of micronucleated cells per 2000 Mature Erythrocytes
Negative control	0	31	0.1	0.6
Dimethyl glutarate	0.5 mg/L	32	0.4	0.0
	1.0 mg/L	31	0.4	0.3
	2.0 mg/L	32	0.2	0.6
Cyclophosphamide	20 mg/kg	27	5.9 (p<0.001)	0.0

Conclusions:

Interpretation of results (migrated information): negative
Dimethyl glutarate does not show any evidence of causing chromosomal damage or bone marrow cell toxicity when administered by whole body inhalation exposure.

Executive summary:

Dimethyl glutarate was administered to F344 rats by whole body inhalation to measure the potential to form micronuclei in bone marrow. Animals were exposed to 0, 0.5. 1.0 and 2.0 mg/l ( analyzed as 0, 0.545, 0.807, and 2.298 mg/L) for 6 hours per day for 2 consecutive days. A positive control group received 20 mg/kg of cyclophophamide by gastric intubation. The were some incidence of weight loss but not considered significant as well as clinical sign (lethargy, partially closed eyes, piloerection, smacking mouth, brown staining of snout and jaws, and oily fur).

Bone marrow smears were obtained 24 hours after exposure from treated and control animals. Smears were were evaluated for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes.

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observe in dimethyl glutarate-treated animals.

Dimethyl glutarate does not show any evidence of causing chromosomal damage or bone marrow cell toxicity when administered by whole body inhalation exposure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

.

Justification for classification or non-classification

No data are available on the reaction mass of dibutyl adipate and glutarate. Studies available on components or analogues: key studies- Ames assay on dibutyl adipate, mammalian cell genoxtoxicity on dimethyl glutarate. Supporting studies - Ames assay on mix of dimethyl esters. Weight of Evidence studies - in vitro clastogenicity assay on dibutyl adipate, in vitro clastogenicity assay on reaction mass of dimethyl esters, in vivo clastogenicity assays on dimethyl glutarate and reaction mass of methyl esters

Overall, based on the available read across information the genetic toxicity of dibasic ester blend is considered to be negative and therefore no classification is warranted according to the classification criteria of Annex VI Directive 67/548/EEC or UN/EU GHS.