Reaction mass of diisobutyl adipate and diisobutyl glutarate and diisobutyl succinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

95481-62-2

Cas Number:

95481-62-2

IUPAC Name:

95481-62-2

Details on test material:

- Name of test material (as cited in study report): DBE

Method

Species / strain

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

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: in presence of S9

Controls 2

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).

Results and discussion

Test results

Test results 1

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

Test results 2

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.