8,9,10-trinorborna-2,5-diene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2017 - February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

8,9,10-trinorborna-2,5-diene

EC Number:

204-472-0

EC Name:

8,9,10-trinorborna-2,5-diene

Cas Number:

121-46-0

Molecular formula:

C7H8

IUPAC Name:

bicyclo[2.2.1]hepta-2,5-diene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch number of test material: Air Liquide Electronics Materials. Batch 1590035136 / 1590035137
- Purity: 100% w/w
- Production date: 10.08.2017
- Expiry date: 16.08.2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material and during storage: stable

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation system: S9 Mix

Type and composition of metabolic activation system:
- source of S9:
S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOX TM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA)

- method of preparation of S9 mix:
S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate.
S9 fraction: 10%
MgCl2-6H2O: 8 mM
KCl: 33 mM
Glucose-6-Phosphate Na2: 5 mM
NADP Na2: 4 mM
Phosphate buffer pH 7.4: 0.1 M

- concentration or volume of S9 mix and S9 in the final culture medium:
Preparation of S9-mix 10 % (v/v)

- Sterility controls of S9:
Test item and the corresponding dilutions with S9-mix are added to 2 mL of top agar maintained at 45°C, and poured after homogenization on the bottom agar (20 ml) onto a Petri plate (90 mm in diameter) (n = 3). Plates are incubated for 48 - 72 hours at 37°C and then examined. There should be no bacterial growth on any plate.

Test concentrations with justification for top dose:

Concentration of the test item: 5 000, 1 500, 500, 150 et 50 µg/plate

Vehicle / solvent:

- Vehicle/solvent used to solubilize test item: DMSO
- Vehicle/solvent used to solubilize positive controls: acetone ; DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

2-nitrofluorene

sodium azide

other: cis-platinum(ii)diamine dichloride ; 9-Aminoacridine ; 2-Anthramine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: negative control, positive control solvent, positive control, vehicle and five dose tested with and without metabolic activation (-S9-mix) for each strains (5 strains).

ASSAY WITHOUT METABOLIC ACTIVATION
Salmonella Typhimurium strains: for each strain, 0.1 mL (in aqueous or oily vehicle) / 50 µL (in non-aqueous or non-oily vehicule as DMSO) of the bacterial suspension containing 1-9 x10^9 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar, maintained supercooled at 45°C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM).

Escherichia coli strain : in a test tube 0.1 mL (in aqueous or oily vehicle) / 50 µL (in non-aqueous or non-oily vehicule as DMSO) of the bacterial suspension containing 1-9 x 10^9 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled in 45°C containing 5% (v/v) of nutrient broth n°2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/mL.
Plates are incubated at 37°C over a 48-72 hour period. The number of revertant colonies per plate is counted.
Moreover the following controls are carried out:
- Negative controls: (i) absolute negative control containing no test item corresponding to the spontaneous reversion rate, (i) solvent used to solubilize positive controls ; Acetone ,DMSO, NaCl 0.15M
- Vehicle used to solubilize test item ; DMSO
- Positive control.

ASSAY WITH METABOLIC ACTIVATION
Two protocols can be used:
• either a standard plate incorporation method where the protocol is similar to that described above, except that, 500 µL of S9-mix fraction is quickly added, before pouring the mixture onto the plates ;
• or the pre-incubation assay where the solution of the test item solution with the test strain, and 500 µL of S9-mix fraction are preincubated with shaking for 30 min., at 37°C prior to mixing with the overlay agar and pouring onto the minimal agar plate.
This method is known to increase the detection sensitivity of a number of promutagens like alkaloids, aliphatic N-Nitroso compounds (OECD no 471).
For the first assay direct incorporation method is used.

PRELIMINARY CYTOTOXICITY TESTING (STRAIN TA100)
In a test tube 0.1 mL of the bacterial suspension (1-9 x 10^3 bacteria/mL) and 0.1 mL (in aqueous or oily vehicle) / 50 µL (in non-aqueous or non-oily vehicule as DMSO) of the stock solution and dilutions, are successively added to 2 mL of top agar at 45°C, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube is poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration are incubated for 48-72 hours at 370 C, and the colonies counted. A negative control containing the blank alone is run in parallel.
In case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less. The precipitate, if present, should not interfere with the scoring. The following four dilutions studied are distributed according to a semi-logarithmic progression.

Evaluation criteria:

Ensure that the criteria of validity of the study are well respected namely:
• the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
• the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
• the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
• the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
• Negative and positive values should not show significant difference with the historical values of the laboratory ± 2 standard deviations).

Results and discussion

Test results

Test results 1

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

Positive controls validity:

valid

Test results 2

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

Positive controls validity:

valid

Test results 3

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

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

• There is no significant difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (without and with metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.
• One can observe in presence of the higher doses tested 5 000 µg/plate and 1 500 µg/plate with metabolic activation and pre-incubation a thinning of the bacterial lawn.
• There is no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (5 000, 1 500, 500, 150 et 50 µg/plate) without and with metabolic activation in (Salmonella lyphimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA) (pKM 101).
• Results are confirmed in an independent experiment.

Any other information on results incl. tables

TA 1535 — Assay no 1 of mutagenic activity

without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	8	8	11	9.00	1.73	-
Positive control solvent	5 µL	9	12	16	12.33	3.51	-
Positive control : Sodium azide	5µg in 5 µL	1213	1226	1198	1212.33	14.01	98.30
Vehicle	50 µL	8	11	14	11.00	3.00	-
Solution of BCHD	5000 µg	14	13	6	11.00	4.36	1.00
	1500 µg	15	13	16	14.67	1.53	1.33
	500 µg	12	12	9	11.00	1.73	1.00
	150 µg	12	14	9	11.67	2.52	1.06
	50 µg	9	15	7	10.33	4.16	0.94

 

with metabolic activation (10 % S9-mix) — without pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	7	13	12	10.67	3.21	-
Positive control solvent	5 µL	13	10	7	10.00	3.00	-
Positive control : Sodium azide	5µg in 5 µL	197	216	180	197.67	18.01	19.77
Vehicle	50 µL	13	16	14	14.33	1.53	-
Solution of BCHD	5000 µg	10	18	11	13.00	4.36	0.91
	1500 µg	11	11	11	11.00	0.00	0.77
	500 µg	13	10	15	12.67	2.52	0.88
	150 µg	14	12	13	13.00	1.00	0.91
	50 µg	15	11	15	13.67	2.31	0.95

 

 

TA 1535 — Assay n02 of mutagenic activity

without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	10	9	7	8.67	1.53	-
Positive control solvent	5 µL	9	13	8	10.00	2.65	-
Positive control : Sodium azide	5µg in 5 µL	924	937	917	926.00	10.15	92.60
Vehicle	50 µL	5	12	6	7.67	3.79	-
Solution of BCHD	5000 µg	8	6	10	8.00	2.00	1.04
	1500 µg	14	7	9	10.00	3.61	1.30
	500 µg	15	11	12	12.67	2.08	1.65
	150 µg	11	9	12	10.67	1.53	1.39
	50 µg	9	11	10	10.00	1.00	1.30

 

with metabolic activation (10 % S9-mix) — with pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	10	10	11	10.33	0.58	-
Positive control solvent	5 µL	14	11	12	12.33	1.53	-
Positive control : Sodium azide	5µg in 5 µL	62	79	74	71.67	8.74	5.81
Vehicle	50 µL	8	8	6	7.33	1.15	-
Solution of BCHD	5000 µg	2	5	3	3.33	1.53	0.45
	1500 µg	8	5	3	5.33	2.52	0.73
	500 µg	11	5	17	11.00	6.00	1.50
	150 µg	8	9	11	9.33	1.53	1.27
	50 µg	7	8	11	8.67	2.08	1.18

 

 

TA 1537 -Assay no 1 of mutagenic activity

without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	10	10	6	8.67	2.31	-
Positive control solvent	5 µL	10	8	7	8.33	1.53	-
Positive control : Sodium azide	5µg in 5 µL	1810	1214	1847	1623.67	355.26	194.84
Vehicle	50 µL	6	7	8	7.00	1.00	-
Solution of BCHD	5000 µg	6	6	5	5.67	0.58	0.81
	1500 µg	5	8	9	7.33	2.08	1.05
	500 µg	7	8	7	7.33	0.58	1.05
	150 µg	9	10	5	8.00	2.65	1.14
	50 µg	10	10	4	8.00	3.46	1.14

 

with metabolic activation (10 % S9-mix) — without pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	18	17	20	18.33	1.53	-
Positive control solvent	5 µL	17	19	22	19.33	2.52	-
Positive control : Sodium azide	5µg in 5 µL	94	99	92	95.00	3.61	4.91
Vehicle	50 µL	13		18	15.00	2.65	-
Solution of BCHD	5000 µg	13	17	19	16.33	3.06	1.09
	1500 µg	12	11	12	11.67	0.58	0.78
	500 µg	17	14	14	15.00	1.73	1.00
	150 µg	15	13	18	15.33	2.52	1.02
	50 µg	20	17	11	16.00	4.58	1.07

 

 

TA 1537 — Assay n02 of mutagenic activity

without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	5	5	4	4.67	0.58	-
Positive control solvent	5 µL	3	5	11	6.33	4.16	-
Positive control : Sodium azide	5µg in 5 µL	368	486	421	425.00	59.10	67.11
Vehicle	50 µL	7	9	12	9.33	2.52	-
Solution of BCHD	5000 µg	14	5	10	9.67	4.51	1.04
	1500 µg	17	9	6	10.67	5.69	1.114
	500 µg	11	18	5	11.33	6.51	1.21
	150 µg	8	10	17	11.67	4.73	1.25
	50 µg	6	17	7	10.00	6.08	1.07

 

with metabolic activation (10 % S9-mix) — with pre-incubation

 

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	18	12	15	15.00	3.00	-
Positive control solvent	5 µL	17	9	23	16.33	7.02	-
Positive control : Sodium azide	5µg in 5 µL	31	88	40	53.00	30.64	3.24
Vehicle	50 µL	10	16	17	14.33	3.79	-
Solution of BCHD	5000 µg	5	2	3	3.33	1.53	0.23
	1500 µg	7	5	8	6.67	1.53	0.47
	500 µg	9	10	11	10.00	1.00	0.70
	150 µg	9	14	11	11.33	2.52	0.79
	50 µg	13	14	12	13.00	1.00	0.91

 

 

TA 98— Assay no 1 of mutagenic activity

without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	10	19	19	16.00	5.20	-
Positive control solvent	5 µL	19	8	12	13.00	5.57	-
Positive control : Sodium azide	5µg in 5 µL	571	509	700	593.33	97.44	45.64
Vehicle	50 µL	16	12	16	14.67	2.31	-
Solution of BCHD	5000 µg	13	11	16	13.33	2.52	0.91
	1500 µg	15	19	18	17.33	2.08	1.18
	500 µg	15	9	14	12.67	3.21	0.86
	150 µg	9	10	12	10.33	1.53	0.70
	50 µg	16	15	12	14.33	2.08	0.98

 

with metabolic activation (10 % S9-mix) — without pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	24	25	20	23.00	2.65	-
Positive control solvent	5 µL	25	17	19	20.33	4.16	-
Positive control : Sodium azide	5µg in 5 µL	637	682	649	656.00	23.30	32.26
Vehicle	50 µL	22	15	15	17.33	4.04	-
Solution of BCHD	5000 µg	31	24	21	25.33	5.13	1.46
	1500 µg	25	27	16	22.67	5.86	1.31
	500 µg	13	16	17	18.67	3.79	1.08
	150 µg	27	23	23	24.33	2.31	1.40
	50 µg	27	20	17	21.33	5.13	1.23

 

 

TA 98 -Assay n02 of mutagenic activity

without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	11	11	18	13.33	4.04	-
Positive control solvent	5 µL	21	11	16	16.00	5.00	-
Positive control : Sodium azide	5µg in 5 µL	415	578	478	490.33	82.20	30.65
Vehicle	50 µL	24	14	16	18.00	5.29	-
Solution of BCHD	5000 µg	8	20	15	14.33	6.03	0.80
	1500 µg	8	12	18	12.67	5.03	0.70
	500 µg	18	15	27	20.00	6.24	1.11
	150 µg	12	12	18	14.00	3.46	0.78
	50 µg	15	16	13	14.67	1.53	0.81

 

with metabolic activation (10 % S9-mix) — with pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	21	34	23	26.00	7.00	-
Positive control solvent	5 µL	30	32	21	27.67	5.86	-
Positive control : Sodium azide	5µg in 5 µL	189	188	244	207.00	32.05	7.48
Vehicle	50 µL	11	21	29	20.33	9.02	-
Solution of BCHD	5000 µg	9	8	5	7.33	2.08	0.36
	1500 µg	11	9	15	11.67	3.06	0.57
	500 µg	25	24	26	25.00	1.00	1.23
	150 µg	25	15	27	22.33	6.43	1.10
	50 µg	15	22	27	21.33	6.03	1.05

 

 

TA 100— Assay n o 1 of mutagenic activity

without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	64	70	68	67.33	3.06	-
Positive control solvent	5 µL	61	77	73	70.33	8.33	-
Positive control : Sodium azide	5µg in 5 µL	1560	1466	1282	1436.00	141.41	20.42
Vehicle	50 µL	62	74	70	68.67	6.11	-
Solution of BCHD	5000 µg	64	60	76	66.67	8.33	0.97
	1500 µg	50	63	61	58.00	7.00	0.84
	500 µg	56	59	53	56.00	3.00	0.82
	150 µg	53	42	56	50.33	7.37	0.73
	50 µg	56	54	52	54.00	2.00	0.79

 

with metabolic activation (10 % S9-mix) — without pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	63	73	85	73.67	11.02	-
Positive control solvent	5 µL	63	74	81	72.67	9.07	-
Positive control : Sodium azide	5µg in 5 µL	1202	1074	1176	1150.67	67.66	15.83
Vehicle	50 µL	78	78	77	77.67	0.58	-
Solution of BCHD	5000 µg	68	76	79	74.33	5.69	0.96
	1500 µg	71	63	73	69.00	5.29	0.89
	500 µg	68	71	74	71.00	3.00	0.91
	150 µg	89	65	79	77.67	12.06	1.00
	50 µg	63	64	74	67.00	6.08	0.86

 

 

TA 100 Assay no2 of mutagenic activity

without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	41	47	55	47.67	7.02
Positive control solvent	5 µL	57	54	76	62.33	11.93
Positive control : Sodium azide	5µg in 5 µL	1101	1330	1520	1317.00	209.80	21.13
Vehicle	50 µL	52	44	45	47.00	4.36
Solution of BCHD	5000 µg	54	49	49	50.67	2.89	1.08
	1500 µg	49	45	46	46.67	2.08	0.99
	500 µg	51	61	58	56.67	5.13	1.21
	150 µg	56	43	51	50.00	6.56	1.06
	50 µg	64	54	70	62.67	8.08	133

 

with metabolic activation (10 % S9-mix) — with pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	76	88	58	74.00	15.10	-
Positive control solvent	5 µL	58	61	59	59.33	1.53	-
Positive control : Sodium azide	5µg in 5 µL	367	375	410	384.00	22.87	6.47
Vehicle	50 µL	51	65	63	59.67	7.57	-
Solution of BCHD	5000 µg	74	54	42	56.67	16.17	0.95
	1500 µg	44	39	41	41.33	2.52	0.69
	500 µg	67	62	71	66.67	4.51	1.12
	150 µg	60	53	54	55.67	3.79	0.93
	50 µg	58	62	56	58.67	3.06	0.98

 

 

E. COLI— Assay no 1 of mutagenic activity

without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	165	166	159	163.33	3.79	-
Positive control solvent	5 µL	163	160	161	161.33	1.53	-
Positive control : Sodium azide	5µg in 5 µL	492	479	483	484.67	6.66	3.00
Vehicle	50 µL	161	162	157	160.00	2.65	-
Solution of BCHD	5000 µg	161	159	168	162.67	4.73	1.02
	1500 µg	150	167	151	156.00	9.54	0.98
	500 µg	166	152	152	156.67	8.08	0.98
	150 µg	155	163	153	157.00	5.29	0.98
	50 µg	154	155	164	157.67	5.51	0.99

 

with metabolic activation (10 % S9-mix) — without pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	194	176	163	177.67	15.57	-
Positive control solvent	5 µL	190	178	163	177.00	13.53	-
Positive control : Sodium azide	5µg in 5 µL	576	624	658	619.33	41.20	3.50
Vehicle	50 µL	174	167	183	174.67	8.02	-
Solution of BCHD	5000 µg	172	160	171	167.67	6.66	0.96
	1500 µg	161	179	202	180.67	20.55	1.03
	500 µg	178	183	183	181.33	2.89	1.04
	150 µg	194	216	194	201.33	12.70	1.15
	50 µg	177	205	165	182.33	20.53	1.04

 

 

E. COLI— Assay no 2 of mutagenic activity

without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	130	123	124	125.67	3.79	-
Positive control solvent	5 µL	120	135	123	126.00	7.94	-
Positive control : Sodium azide	5µg in 5 µL	424	521	475	473.33	48.52	3.76
Vehicle	50 µL	148	131	123	134.00	12.77	-
Solution of BCHD	5000 µg	108	180	158	148.67	36.90	1.11
	1500 µg	116	114	126	118.67	6.43	0.89
	500 µg	121	129	124	124.67	4.04	0.93
	150 µg	118	115	129	120.67	7.37	0.90
	50 µg	130	114	114	119.33	9.24	0.89

 

with metabolic activation (10 % S9-mix) — with pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard	R
		n^o1	n^o2	n^o3
Negative control	100 µL	191	189	173	184.33	9.87	-
Positive control solvent	5 µL	153	171	165	163.00	9.17	-
Positive control : Sodium azide	5µg in 5 µL	575	591	610	592.00	17.52	3.63
Vehicle	50 µL	167	162	163	164.00	2.65	-
Solution of BCHD	5000 µg	35	93	51	59.67	29.96	0.36
	1500 µg	80	110	118	102.67	20.03	0.63
	500 µg	157	169	162	162.61	6.03	0.99
	150 µg	183	162	159	168.00	13.08	1.02
	50 µg	186	171	167	174.67	10.02	1.07

 

 

Applicant's summary and conclusion

Conclusions:

Doses (5 000, 1 500, 500, 150 and 50 µg/plate) prepared from solutions of the test item BCHD-Bicyclo 2,2,1-Hepta 2,5 diene (or BCHD) provided by AIR LIQUIDE ELECTRONICS MATERIALS (Batches 1590035136 & 1590035137), do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA1537, TA98, TA 100 and in Escherichia coli WP2(uvrA3 (pKM 101) without, or with metabolic activation, according to the OECD Guidelines n°471.
Based on this Ames test, the substance BCHD is considered not genotoxic.

Executive summary:

A bacterial reverse mutation test was performed using  "Salmonella typhimurium his-" and "Escherichia coli" WP2(uvrA-)(pKM101) according to OECD guideline n^o 471.

Solutions have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out

For assay n°1, various concentrations of solution were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

For assay n°2, various concentrations of solution were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental "historical" values obtained in the laboratory.

These results validate the two tests.

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentration of the test item (5 000, 1 500, 500, 150 et 50 µg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA ) (pKM 101).