Dioctyl maleate, branched

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The key studies were conducted to internationally recognised testing guidelines and with GLP certification.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2017 - 20 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Bernel Ester DCM (CAS number 85566-63-8), batch number P6978, was a clear liquid. It was received on 07 September 2017 and stored at 15-25°C protected from light. Purity was stated as 92.78% (assumed 100% for testing) and the expiry date was given as 06 September 2018, see Certificate of Analysis. The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.
Preliminary solubility data indicated that Bernel Ester DCM was soluble in ethanol at concentrations equivalent to at least 100 mg/mL. A maximum concentration of 5000 µg/plate was selected for Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997). A maximum concentration of 5000 µg/plate was also selected for Experiment 2.
Test article stock solutions were prepared by formulating Bernel Ester DCM under subdued lighting in ethanol with the aid of vortex mixing, to give the maximum required treatment concentration. Subsequent dilutions were made using ethanol. The test article solutions were protected from light and used within approximately 6 hours of initial formulation.

Target gene:

Histidine locus.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Mutation Experiment 1 (S-9+-): 5, 16, 50, 160, 500, 1600, 5000 µg/plate
Mutation Experiment 2 (S-9+-): 160, 320, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Test System
The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls, in the absence or presence of S-9 mix.

Mutation Experiments
Bernel Ester DCM was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45±1°C:
• 0.1 mL bacterial culture
• 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
• 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony Enumeration).
As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.
Volume additions for the Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (ethanol) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

Toxicity Assessment
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Colony Enumeration
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as precipitation, contamination or bubbles or splits in the agar affected the accuracy of the automated counter.

Analysis of Results
Treatment of Data
Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges. Data were considered acceptable if the vehicle control counts fell within the calculated historical control ranges and the positive control plate counts were comparable with the historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate). However, adequate interpretation of biological relevance was of critical importance.

Acceptance Criteria
The assay was to be considered valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges
2. The positive control chemicals induced increases in revertant numbers of ≥1.5 fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

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

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

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

Positive controls validity:

valid

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

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:

Toxicity, Solubility and Concentration Selection
Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Bernel Ester DCM at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments evidence of toxicity in the form of a slight thinning of the background bacterial lawn was observed at 5000 µg/plate in strain TA98 in the absence and presence of S-9 and strain TA98 in the presence of S-9 only.
Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160-5000 µg/plate, in order to examine more closely those concentrations of Bernel Ester DCM approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments no evidence of toxicity was observed.
Precipitation was observed on the test plates at concentrations of 5000 µg/plate.

Data Acceptability and Validity
The individual mutagenicity plate counts were averaged to give mean values, which are presented in Section 8. From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges. The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3 fold (in strains TA1535 and TA1537) the concurrent vehicle controls confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

Mutation
Following Bernel Ester DCM treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Bernel Ester DCM mutagenic activity in this assay system.

Conclusions:

It was concluded that Bernel Ester DCM did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Bernel Ester DCM was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

All Bernel Ester DCM treatments in this study were performed using formulations prepared in ethanol.

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Bernel Ester DCM at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments evidence of toxicity was observed at 5000 µg/plate in strain TA98 in the absence and presence of S-9 and strain TA98 in the presence of S-9 only.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160-5000 µg/plate, in order to examine more closely those concentrations of Bernel Ester DCM approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre‑incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.

Precipitation was observed on all test plates at a concentration of 5000 µg/plate.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell withinacceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following Bernel Ester DCM treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Bernel Ester DCM mutagenic activity in this assay system.

It was concluded that Bernel Ester DCM did not induce mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19/9/2017 to 13/3/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

23 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Bernel Ester DCM (CAS number 85566-63-8), batch number P6978, was a clear liquid, with a molecular weight of 340 g/L. It was received on 07 September 2017 and stored at 15-25°C protected from light. Purity was stated as 92.78% (assumed 100% for testing) and the expiry date was given as 06 September 2018, see Certificate of Analysis. The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human lymphocytes prepared form the pooled blood of two female doners

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Preliminary solubility data indicated that Bernel Ester DCM was soluble in acetone at concentrations up to at least 342.9 mg/mL. The solubility limit in culture medium was in the range of 53.58 to 107.2 µg/mL, as indicated by precipitation at the higher concentration which persisted following 24 hours incubation at 37±1°C. A maximum concentration of 200 µg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to a precipitating treatment concentration (OECD, 2016). Concentrations for the Micronucleus Experiment were selected based on the results of this cytotoxicity Range-Finder Experiment.

Test article stock solutions were prepared by formulating Bernel Ester DCM under subdued lighting in acetone, with the aid of vortex mixing, to give the maximum required treatment concentration. Subsequent dilutions were made using acetone. The test article solutions were protected from light and used within approximately
3.5 hours of initial formulation. The following concentration ranges were tested:
Experiment Treatment Concentration Range Final Concentration Range
(mg/mL) (µg/mL)
Range-Finder
3+21, -S-9 0.07256 to 20.00 0.7256 to 200.0
3+21, +S-9 0.07256 to 20.00 0.7256 to 200.0
24+24, -S-9 0.07256 to 20.00 0.7256 to 200.0
Micronucleus Experiment
3+21, -S-9 0.500 to 6.000 5.000 to 60.00
3+21, +S-9 1.000 to 10.000 10.000 to 100.00
24+24, -S-9 0.500 to 6.000 5.000 to 60.00

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: vinblastine

Evaluation criteria:

Evaluation Criteria
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at very toxic concentrations (Thybaud et al., 2007).

Key result

Species / strain:

lymphocytes: 24+24 hour treatment

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: 24+24 hour treatment

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: 3+21 hour treatment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

RESULTS

Selection of Concentrations for Micronucleus Analysis

The results of the RI determinations from the cytotoxicity Range-Finder Experiment were as follows:

Text Table 1: Data for 3+21 Hour Treatments -S-9, Range-Finder - Female Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	35	158	7	200	0.86
	B	45	148	7	200	0.81
	Total	80	306	14	400	0.84	-
UTC	A	55	140	5	200	0.75	-
0.7256	A	NSc	-	-	-	-	-
1.209	A	NSc	-	-	-	-	-
2.016	A	NSc	-	-	-	-	-
3.359	A	NSc	-	-	-	-	-
5.599	A	43	148	9	200	0.83	1
9.331	A	47	147	6	200	0.80	5
15.55	A	46	147	7	200	0.81	4
25.92	A	78	122	0	200	0.61	27
43.20	A	172	28	0	200	0.14	83
72.00	A	194	6	0	200	0.03	96 P
120.0	A	184	16	0	200	0.08	90 P
200.0	A	186	14	0	200	0.07	92 P

 

Text Table 2: Data for 3+21 Hour Treatments +S-9, Range-Finder - Female Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	35	152	13	200	0.89
	B	30	155	15	200	0.93
	Total	65	307	28	400	0.91	-
UTC	A	30	160	10	200	0.90	-
0.7256	A	NSc	-	-	-	-	-
1.209	A	NSc	-	-	-	-	-
2.016	A	NSc	-	-	-	-	-
3.359	A	NSc	-	-	-	-	-
5.599	A	28	153	19	200	0.96	0
9.331	A	39	142	19	200	0.90	1
15.55	A	40	152	8	200	0.84	7
25.92	A	44	148	8	200	0.82	10
43.20	A	56	141	3	200	0.74	19
72.00	A	159	40	1	200	0.21	77 P
120.0	A	193	7	0	200	0.04	96 P
200.0	A	194	6	0	200	0.03	97 P

UTC = Untreated control                   NSc = Not scored               P = Precipitation observed at treatment

Mono = Mononucleate                      Bi = Binucleate                  Multi = Multinucleate

RI = Replication index

 

Text Table 3: Data for 24+24 Hour Treatments -S-9, Range-Finder - Female Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	18	155	27	200	1.05
	B	19	155	26	200	1.04
	Total	37	310	53	400	1.04	-
UTC	A	18	142	40	200	1.11	-
0.7256	A	14	155	31	200	1.09	0
1.209	A	19	148	33	200	1.07	0
2.016	A	23	155	22	200	1.00	4
3.359	A	25	150	25	200	1.00	4
5.599	A	38	154	22	214	0.93	11
9.331	A	27	142	31	200	1.02	2
15.55	A	61	129	10	200	0.75	28
25.92	A	79	116	5	200	0.63	39
43.20	A	178	22	0	200	0.11	89
72.00	A	189	11	0	200	0.06	95P
120.0	A	NE	-	-	-	-	-P
200.0	A	NE	-	-	-	-	-P

UTC = Untreated control                    

NE = Not evaluated - no scoreable cells             

P = Precipitation observed at treatment

Mono = Mononucleate                       

Bi = Binucleate                   

Multi = Multinucleate

RI = Replication index 

No marked changes in osmolality or pH were observed at the highest concentration tested (200 µg/mL) as compared to the concurrent vehicle controls (individual data not reported).

The results of the cytotoxicity Range-Finder Experiment were used to select suitable concentrations for the Micronucleus Experiment.

The results of the RI determinations from the Micronucleus Experiment were as follows:

Text Table 4: Data for 3+21 Hour Treatments -S-9, Micronucleus Experiment - Female Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	177	316	7	500	0.66
	B	182	310	8	500	0.65
	C	163	328	9	500	0.69
	D	155	339	6	500	0.70
	Total	677	1293	30	2000	0.68	-
UTC	A	163	317	20	500	0.71
	B	128	353	19	500	0.78
	Total	291	670	39	1000	0.75	- #
5.000	A	189	304	7	500	0.64
	B	136	357	7	500	0.74
	Total	325	661	14	1000	0.69	0 #
10.00	A	186	310	4	500	0.64
	B	171	322	7	500	0.67
	Total	357	632	11	1000	0.65	3
15.00	A	179	316	5	500	0.65
	B	155	340	5	500	0.70
	Total	334	656	10	1000	0.68	0
20.00	A	181	316	3	500	0.64
	B	169	331	0	500	0.66
	Total	350	647	3	1000	0.65	3
25.00	A	243	255	2	500	0.52
	B	204	294	2	500	0.60
	Total	447	549	4	1000	0.56	18 #
27.50	A	260	239	1	500	0.48
	B	235	265	0	500	0.53
	Total	495	504	1	1000	0.51	25
30.00	A	305	194	1	500	0.39
	B	324	175	1	500	0.35
	Total	629	369	2	1000	0.37	45 #
32.50	A	317	183	0	500	0.37
	B	347	153	0	500	0.31
	Total	664	336	0	1000	0.34	50 #
35.00	A	408	92	0	500	0.18
	B	359	141	0	500	0.28
	Total	767	233	0	1000	0.23	66
37.50	A	445	55	0	500	0.11
	B	435	65	0	500	0.13
	Total	880	120	0	1000	0.12	82
40.00	A	471	29	0	500	0.06
	B	441	59	0	500	0.12
	Total	912	88	0	1000	0.09	87
45.00	A	486	14	0	500	0.03
	B	484	16	0	500	0.03
	Total	970	30	0	1000	0.03	96
60.00	A	498	2	0	500	0.00
	B	497	3	0	500	0.01
	Total	995	5	0	1000	0.01	99

Table continued overleaf

  

Text Table 4 Continued: Data for 3+21 Hour Treatments -S-9, Micronucleus Experiment - Female Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
MMC, 0.20	A	220	280	0	500	0.56
	B	240	259	1	500	0.52
	Total	460	539	1	1000	0.54	20
MMC, 0.30	A	285	215	0	500	0.43
	B	259	240	1	500	0.48
	Total	544	455	1	1000	0.46	32 #

UTC = Untreated control                    

Mono = Mononucleate 

Bi = Binucleate 

Multi = Multinucleate

RI = Replication index

 

# Highlighted concentrations selected for analysis

   

Text Table 5: Data for 3+21 Hour Treatments +S-9, Micronucleus Experiment - Female Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	79	372	49	500	0.94
	B	73	392	35	500	0.92
	C	77	388	35	500	0.92
	D	76	393	31	500	0.91
	Total	305	1545	150	2000	0.92	-
UTC	A	130	339	31	500	0.80
	B	96	364	40	500	0.89
	Total	226	703	71	1000	0.85	- #
10.00	A	123	355	22	500	0.80
	B	115	361	24	500	0.82
	Total	238	716	46	1000	0.81	12 #
20.00	A	122	351	27	500	0.81
	B	102	378	20	500	0.84
	Total	224	729	47	1000	0.82	11
30.00	A	138	341	21	500	0.77
	B	138	353	9	500	0.74
	Total	276	694	30	1000	0.75	18
35.00	A	115	369	16	500	0.80
	B	141	342	17	500	0.75
	Total	256	711	33	1000	0.78	16
40.00	A	141	355	4	500	0.73
	B	135	354	11	500	0.75
	Total	276	709	15	1000	0.74	20
45.00	A	177	318	5	500	0.66
	B	177	321	2	500	0.65
	Total	354	639	7	1000	0.65	29 #
50.00	A	192	300	8	500	0.63
	B	147	349	4	500	0.71
	Total	339	649	12	1000	0.67	27
55.00	A	225	274	1	500	0.55
	B	219	280	1	500	0.56
	Total	444	554	2	1000	0.56	40
60.00	A	263	236	1	500	0.48
	B	255	241	4	500	0.50
	Total	518	477	5	1000	0.49	47
65.00	A	316	184	0	500	0.37
	B	289	209	2	500	0.43
	Total	605	393	2	1000	0.40	57 #
70.00	A	388	112	0	500	0.22
	B	423	77	0	500	0.15
	Total	811	189	0	1000	0.19	80
80.00	A	423	77	0	500	0.15
	B	433	67	0	500	0.13
	Total	856	144	0	1000	0.14	84
100.0	A	487	13	0	500	0.03
	B	484	14	0	498	0.03
	Total	971	27	0	998	0.03	97

Table continued overleaf

 

Text Table 5 Continued: Data for 3+21 Hour Treatments +S-9, Micronucleus Experiment - Female Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
CPA, 2.00	A	150	348	2	500	0.70
	B	139	353	8	500	0.74
	Total	289	701	10	1000	0.72	22
CPA, 3.00	A	143	350	7	500	0.73
	B	128	367	5	500	0.75
	Total	271	717	12	1000	0.74	20 #

UTC = Untreated control                    

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

 

# Highlighted concentrations selected for analysis

  

Text Table 6: Data for 24+24 Hour Treatments -S-9, Micronucleus Experiment - Female Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	134	331	35	500	0.80
	B	107	349	44	500	0.87
	C	105	353	42	500	0.87
	D	106	358	36	500	0.86
	Total	452	1391	157	2000	0.85	-
UTC	A	83	377	40	500	0.91
	B	68	367	65	500	0.99
	Total	151	744	105	1000	0.95	-
5.000	A	104	371	25	500	0.84
	B	102	373	25	500	0.85
	Total	206	744	50	1000	0.84	1
10.00	A	97	379	24	500	0.85
	B	71	397	32	500	0.92
	Total	168	776	56	1000	0.89	0
15.00	A	115	361	24	500	0.82
	B	103	368	29	500	0.85
	Total	218	729	53	1000	0.84	2 #
17.50	A	125	362	13	500	0.78
	B	134	357	9	500	0.75
	Total	259	719	22	1000	0.76	10
20.00	A	169	322	9	500	0.68
	B	131	358	11	500	0.76
	Total	300	680	20	1000	0.72	16
22.50	A	162	332	6	500	0.69
	B	179	319	2	500	0.65
	Total	341	651	8	1000	0.67	22 #
25.00	A	259	238	3	500	0.49
	B	265	234	1	500	0.47
	Total	524	472	4	1000	0.48	44
27.50	A	294	203	3	500	0.42
	B	271	227	2	500	0.46
	Total	565	430	5	1000	0.44	48 #
30.00	A	340	160	0	500	0.32
	B	384	115	1	500	0.23
	Total	724	275	1	1000	0.28	68
32.50	A	400	100	0	500	0.20
	B	413	86	1	500	0.18
	Total	813	186	1	1000	0.19	78
35.00	A	451	49	0	500	0.10
	B	404	96	0	500	0.19
	Total	855	145	0	1000	0.15	83
40.00	A	472	28	0	500	0.06
	B	479	21	0	500	0.04
	Total	951	49	0	1000	0.05	94

Table continued overleaf

 

Text Table 6 Continued: Data for 24+24 Hour Treatments -S-9, Micronucleus Experiment - Female Donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
45.00	A	475	25	0	500	0.05
	B	485	15	0	500	0.03
	Total	960	40	0	1000	0.04	95
60.00	A	492	8	0	500	0.02
	B	493	7	0	500	0.01
	Total	985	15	0	1000	0.02	98
VIN, 0.04	A	220	231	49	500	0.66
	B	207	240	53	500	0.69
	Total	427	471	102	1000	0.68	21 #
VIN, 0.06	A	287	168	45	500	0.52
	B	323	139	38	500	0.43
	Total	610	307	83	1000	0.47	45

UTC = Untreated control

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

 

# Highlighted concentrations selected for analysis 

Micronucleus Analysis

Raw Data

The raw data for the observations on the test article plus positive and vehicle controls are retained by Covance Laboratories Ltd. A summary of the number of cells containing micronuclei is given inTable 8.1toTable 8.3.

Validity of Study

The data inTable 8.1toTable 8.6, Section7.3andText Table 4toText Table 6confirm that:

1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures (Table 8.4 to Table 8.6)

2. The frequency of MNBN cells in vehicle controls fell within the normal ranges (Section 7.3)

3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range (Table 8.1 to Table 8.3)

4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest (Text Table 4 to Text Table 6)

5. The maximum concentration analysed under each treatment condition met the criteria specified in Section 4.7.

Analysis of Data

3+21 hour treatment in the absence of S-9

Treatment of cells with Bernel Ester DCM for 3+21 hours in the absence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for three of the four concentrations analysed (Table 8.1andTable 8.4) . A weak but statistically significant increase was observed at the highest concentration analysed (32.5 µg/mL, inducing 50% cytotoxicity). This increase was only marginally above the 95^thpercentile of the observed (normal) range (mean frequency of 1.03% MNBN cells against a range of 0.2 to 1.0% MNBN cells) and fell within the current observed range (0.1 to 1.2% MNBN cells) (Section7.3). Furthermore the increase was noted in a single replicate only at this concentration. 

With the exception of a single replicate at an intermediate concentration (25 µg/mL, giving 18% cytotoxicity) all other treated cultures demonstrated MNBN cell frequencies within the normal range. A statistically significant linear trend (p≤0.05) was observed. The weak but statistically significant increase noted at 32.5 µg/mL was poorly reproduced between replicates and is therefore considered of questionable biological relevance.

No test article related increases in cells with NPBs were observed (data not reported).

3+21 hour treatment in the presence of S-9

Treatment of cells with Bernel Ester DCM for 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed (Table 8.2andTable 8.5). With the exception of a single culture at the lowest concentration analysed (10 µg/mL, giving 12% cytotoxicity) all test article treated cultures exhibited MNBN cell frequency that fell within the normal range (Section7.3). These data indicate a negative result.

No test article related increases in cells with NPBs were observed (data not reported).

24+24 hour treatment in the absence of S-9

Following 24+24 hour treatment in the absence of S-9, an increase in the frequency of MNBN cells, which was significantly (p≤0.05) higher than that observed in the concurrent vehicle controls, was observed at the highest concentration analysed (27.5 µg/mL) (Table 8.3andTable 8.6). The observed increase exceeded the normal range in both replicate cultures and a statistically significant linear trend (p≤0.05) was observed, indicating a positive result under this treatment condition (Section7.3).

No test article related increases in cells with NPBs were observed (data not reported).

Conclusions:

It is concluded that Bernel Ester DCM showed evidence of micronucleus induction in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of an Aroclor-induced rat liver metabolic activation system (S-9). In the same test system, Bernel Ester DCM did not induce biologically relevant increases in micronuclei following 3+21 hour treatment in the absence and presence of S-9. Sporadic increases were noted following 3+21 hour treatment in the absence of S-9 but there was poor reproducibility between replicates and therefore this observation was considered of questionable biological relevance. Maximum concentrations analysed were limited by toxicity under all treatment conditions (in line with current regulatory test guidelines for the in vitro micronucleus assay).

Executive summary:

Bernel Ester DCM was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in acetone and the highest concentrations tested in the Micronucleus Experiment, (limited by toxicity) were determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Bernel Ester DCM on the replication index (RI). Micronuclei were analysed at three or four concentrations and a summary of the data is presented in the following table:

Treatment	Concentration (mg/mL)	Cytotoxicity (%)^$	Mean MNBN Cell Frequency (%)	Historical Control Range (%)^#	Statistical Significance
3+21 hour -S-9	Vehicle^a	-	0.65	0.20 – 1.00	-
	UTC	-	0.85		-
	5.000	0	0.55		NS
	25.00	18	0.85		NS
	30.00	45	0.80		NS
	32.50	50	1.03		p≤0.05
	*MMC, 0.30	32	5.00		p≤0.001
3+21 hour +S-9	Vehicle^a	-	0.83	0.20 – 1.07	-
	UTC	-	0.60		-
	10.00	12	0.98		NS
	45.00	29	0.68		NS
	65.00	57	0.70		NS
	*CPA, 3.00	20	2.20		p≤0.001
24+24 hour -S-9	Vehicle^a	-	0.65	0.10 – 0.90	-
	15.00	2	0.55		NS
	22.50	22	0.45		NS
	27.50	48	1.20		p≤0.05
	*VIN, 0.04	21	3.10		p≤0.001

Appropriate negative (vehicle and untreated) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in these cultures fell within the 95^thpercentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.

All acceptance criteria were considered met and the study was therefore accepted as valid.

3+21 hour treatment in the absence of S-9

Treatment of cells withBernel EsterDCMfor 3+21 hours in the absence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for three of the four concentrationsanalysed. A weak but statistically significant increase was observed at the highest concentration analysed (32.5 µg/mL, inducing 50% cytotoxicity). This increase was only marginally above the 95^thpercentile of the observed (normal) range (mean frequency of 1.03% MNBN cells against a range of 0.2 to 1.0% MNBN cells) and fell within the current observed range (0.1 to 1.2% MNBN cells). Furthermore the increase was noted in a single replicate only at this concentration.

With the exception of a single replicate at an intermediate concentration (25 µg/mL, giving 18% cytotoxicity) all other treated cultures demonstrated MNBN cell frequencies within the normal range. A statistically significant linear trend (p≤0.05) was observed. The weak but statistically significant increase noted at 32.5 µg/mL was poorly reproduced between replicates and is therefore considered of questionable biological relevance.

3+21 hour treatment in the presence of S-9

Treatment of cells withBernel EsterDCMfor 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for all concentrationsanalysed. With the exception of a single culture at the lowest concentrationanalysed(10 µg/mL, giving 12% cytotoxicity) all test article treated cultures exhibited MNBN cell frequency that fell within the normal range. These data indicate a negative result.

24+24 hour treatment in the absence of S-9

Following 24+24 hour treatment in the absence of S-9, an increase in the frequency of MNBN cells, which was significantly (p≤0.05) higher than that observed in the concurrent vehicle controls, was observed at the highest concentration analysed (27.5 µg/mL). The observed increase exceeded the normal range in both replicate cultures and a statistically significant linear trend (p≤0.05) was observed, indicating a positive result under this treatment condition.

Conclusion

It is concluded thatBernel EsterDCMshowed evidence of micronucleus induction in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of an Aroclor-induced rat liver metabolic activation system (S‑9). In the same test system,Bernel EsterDCMdid not induce biologically relevant increases in micronuclei following 3+21 hour treatment in the absence and presence of S-9. Sporadic increases were noted following 3+21 hour treatment in the absence of S-9 but there was poor reproducibility between replicates and therefore this observation was considered of questionable biological relevance. Maximum concentrations analysed were limited by toxicity under all treatment conditions (in line with current regulatory test guidelines for thein vitromicronucleus assay).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

To be defined

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: awaiting final result

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The registered substance did not induce mutations in the AMES test and the micronucleus study was considered to be inconclusive. It was therefore considered that the registered substance failed did not meet the criteria for classification as a mutagen under the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).