Reaction mass of (1R,4S,4aR,8R,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one and (1S,4R,4aS,8S,8aR)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one and (1R,4S,4aR,8S,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one and (1S,4R,4aS,8R,8aR)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2010-12-06 to 2011-01-11 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study has been conducted according to the standard guideline(s) and its results are acceptable for assessment and classification. The study has not been conducted fully according to GLP. It was conducted in a facility operating to GLP within the UK national GLP monitoring programme and it was Quality Assurance audited to verify the integrity of the data and to ensure the report accurately reflects the data. The analytical characterisation of the test material has been performed according to GLP. As a consequence, the overall reliability rating (according to Klimisch et al.) is considered to be between "reliable without restrictions" and "reliable with restrictions", but closer to "reliable without restrictions". Thus an overall Klimisch rating of 1 (reliable without restrictions) has been assigned.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Principles of method if other than guideline:

- The study was based on the in vitro technique described by Ames and his co-workers and Garner et al in which mutagenic activity is assessed by exposing histidine and tryptophan auxotrophs of Salmonella typhimurium and Escherichia coli to various concentrations of a test item.
- The dose range for the main experiment was determined in a preliminary toxicity assay.
- In the main experiment, six strains were tested (TA98, TA100, TA1535, TA1537, WP2uvrApKM101, WP2pKM101
- Each bacterial strain was treated with each concentration of test item in triplicate both with and without S9-mix in Experiment I and with S9-mix only in Experiment II.
- The plate incorporation method was used for Experiment I and the preincubation method for Experiment II.

GLP compliance:

no

Remarks:

Conducted in a facility operating to GLP within the UK national GLP monitoring programme. Audited by Quality Assurance to verify the integrity of the data and to ensure the report accurately reflects the data. Analysis of test item according to GLP.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (1R,4S,4aR,8R,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one and (1S,4R,4aS,8S,8aR)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one and (1R,4S,4aR,8S,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one and (1S,4R,4aS,8R,8aR)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one

EC Number:

700-770-4

Molecular formula:

C12H14Cl2O2

IUPAC Name:

Reaction mass of (1R,4S,4aR,8R,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one and (1S,4R,4aS,8S,8aR)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one and (1R,4S,4aR,8S,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one and (1S,4R,4aS,8R,8aR)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one

Details on test material:

- Physical state: Light beige powder
- Stability under test conditions: not reported
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

Histidine locus of the genom of the bacterium for Salmonella typhimurium
Tryptophan locus of the genom of the bacterium for Escherichia coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: E. coli WP2 strain used: E. coli WP2 pKM101

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolising system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Preliminary toxicity test, with and without S9: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Experiment I, without S9: 15, 50, 150, 500, 1500, 5000 µg/plate
Experiment I, with S9: 15, 50, 150, 500, 1500, 5000 µg/plate
Experiment II, with S9: 5, 15, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: dimethyl sulphoxide (for vehicle controls, positive controls, test item formulation)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Without S9: N-ethyl-N'-nitro-N-nitrosoguanidine (TA100, TA1535, WP2uvrA pKM101 , WP2pKM101), 4-nitroquinoline-1-oxide (TA98), 9-aminoacridine (TA1537). With S9: 2-aminoanthracene (TA100, TA1535, WP2uvrA pKM101 , WP2pKM101, TA1537), benzo(a)pyrene (TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation)
Experiment II: preincubation

DURATION
- Preincubation period: 60 minutes (37°C, Experiment II only)
- Exposure duration: incubation at 37°C for approx. 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY:
- Method (preliminary toxicity test):
concentrations tested: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate;
after approx. 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 μg/plate because of excessive test item precipitation.

MEASUREMENT OF FREQUENCY OF REVERTANT COLONIES:
by means of a Domino colony counter or (in case of excessive test item precipitation) by manual counts

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Mainly at the highest dose level(s). For details see "ADDITIONAL INFORMATION ON CYTOTOXICITY" under "Additional information on results".

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Mainly at the highest dose level(s). For details see "ADDITIONAL INFORMATION ON CYTOTOXICITY" under "Additional information on results".

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (fibrous in appearance) was noted at 5000 µg/plate in both experiments. This observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: Strain TA100 and WP2uvrApKM101 were treated with concentrations from 0 to 5000 µg/plate with and without metabilic activation. The results are presented in "Any other information on results incl. tables".

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test item was toxic to both of the strains of bacteria used (TA100 (presence and absence of S9-mix) and WP2uvrApKM101 (absence of S9-mix only)) at 5000 μg/plate (the test item formulation and S9-mix used in this experiment were both shown to be sterile).
In the first experiment (plate incorporation methodology) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of S9-mix at 5000 µg/plate. In the presence of S9-mix, weakened bacterial background lawns were noted for TA100, TA98 and TA1537 at 5000 µg/plate with substantial reductions in revertant colony frequency noted at the same dose level for TA1535, WP2uvrApKM101 and WP2pKM101. In the second experiment (pre-incubation methodology) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 1500 µg/plate (TA100) and at 5000 µg/plate (TA1535, TA98 and TA1537). There was no visible reduction in the viability of the bacterial background lawns of Escherichia coli strains WP2uvrApKM101 and WP2pKM101, however slight reductions in colony frequency were noted.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Increases in the frequency of revertant colonies

No toxicologically significant increases in the frequency of revertant colonies, in excess of two‑fold greater than the concurrent solvent controls, were recorded for any of the bacterial strains, for any dose level of the test item, either with or without metabolic activation.

 

 

Preliminary Toxicity Test

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	87	93	84	95	90	86	84	78	95	85	0 VP
+	TA100	102	106	113	120	102	121	122	103	113	87	0 VP
-	WP2uvrA pKM101	140	155	158	154	135	134	144	151	134	137	85 SP
+	WP2uvrA pKM101	173	166	161	201	173	170	177	180	188	184	130 P

S            Sparse bacterial background lawn

V            Very weak bacterial background lawn

P            Precipitate

 

 

Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 13 December 2010						To: 16 December 2010
With or Without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type								Frameshift type
		TA100		TA1535		WP2uvrA pKM101		WP2pKM101		TA98		TA1537
-	0	102 107 103	(104) 2.6#	26 20 27	(24) 3.8	203 197 198	(199) 3.2	81 81 99	(87) 10.4	22 22 23	(22) 0.6	11 7 16	(11) 4.5
-	15	82 90 96	(89) 7.0 0.9	20 27 20	(22) 4.0 0.9	177 206 197	(193) 14.8 1.0	103 92 97	(97) 5.5 1.1	15 15 18	(16) 1.7 0.7	12 14 12	(13) 1.2 1.2
-	50	101 101 93	(98) 4.6 0.9	15 26 20	(20) 5.5 0.8	195 214 189	(199) 13.1 1.0	85 113 81	(93) 17.4 1.1	24 20 16	(20) 4.0 0.9	14 12 15	(14) 1.5 1.3
-	150	98 89 106	(98) 8.5 0.9	26 26 23	(25) 1.7 1.0	186 188 201	(192) 8.1 1.0	89 101 82	(91) 9.6 1.0	19 18 20	(19) 1.0 0.9	11 11 11	(11) 0.0 1.0
-	500	86 92 96	(91) 5.0 0.9	29 24 24	(26) 2.9 1.1	200 179 198	(192) 11.6 1.0	81 81 78	(80) 1.7 0.9	21 16 21	(19) 2.9 0.9	14 15 11	(13) 2.1 1.2
-	1500	117 75 101	(98) 21.2 0.9	24 23 29	(25) 3.2 1.0	190 176 217	(194) 20.8 1.0	67 74 75	(72) 4.4 0.8	15 18 16	(16) 1.5 0.7	11 8 9	(9) 1.5 0.8
-	5000	69 SP 78 SP 81 SP	(76) 6.2 0.7	0 VP 0 VP 0 VP	(0) 0.0 0.0	126 SP 172 SP 77 SP	(125) 47.5 0.6	56 SP 31 SP 45 SP	(44) 12.5 0.5	0 VP 0 VP 0 VP	(0) 0.0 0.0	0 VP 0 VP 0 VP	(0) 0.0 0.0
Positive controls	Name	ENNG		ENNG		ENNG		ENNG		4NQO		9AA
S9-Mix -	Concentration (μg/plate)	3		5		0.5		2		0.2		80
	No. colonies per plate	449 346 454	(416) 61.0 4.0	1212 1052 1290	(1185) 121.3 49.4	1486 1709 1784	(1660) 155.0 8.3	1784 1597 1730	(1704) 96.2 19.6	140 150 157	(149) 8.5 6.8	2203 2882 2046	(2377) 444.3 216.1

ENNG   N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO   4-Nitroquinoline-1-oxide

9AA     9-Aminoacridine

S           Sparse bacterial background lawn

V           Very weak bacterial background lawn

P           Precipitate

0.0        Fold increase over concurrent vehicle control

#        Standard deviation

 

 

Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 13 December 2010						To: 16 December 2010
With or Without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type								Frameshift type
		TA100		TA1535		WP2uvrA pKM101		WP2pKM101		TA98		TA1537
+	0	103 91 99	(98) 6.1#	16 15 11	(14) 2.6	252 286 231	(256) 27.8	135 130 128	(131) 3.6	30 23 24	(26) 3.8	14 11 15	(13) 2.1
+	15	90 82 93	(88) 5.7 0.9	15 12 12	(13) 1.7 0.9	242 227 210	(226) 16.0 0.9	98 123 136	(119) 19.3 0.9	27 21 30	(26) 4.6 1.0	14 16 14	(15) 1.2 1.2
+	50	79 80 74	(78) 3.2 0.8	10 14 12	(12) 2.0 0.9	222 217 213	(217) 4.5 0.8	115 133 130	(126) 9.6 1.0	33 22 19	(25) 7.4 1.0	14 8 13	(12) 3.2 0.9
+	150	91 74 84	(83) 8.5 0.8	16 9 23	(16) 7.0 1.1	232 206 269	(236) 31.7 0.9	118 122 115	(118) 3.5 0.9	22 23 25	(23) 1.5 0.9	12 12 13	(12) 0.6 0.9
+	500	86 104 80	(90) 12.5 0.9	16 15 12	(14) 2.1 1.0	229 236 253	(239) 12.3 0.9	114 115 121	(117) 3.8 0.9	29 22 23	(25) 3.8 1.0	10 13 14	(12) 2.1 0.9
+	1500	95 86 99	(93) 6.7 0.9	3 8 3	(5) 2.9 0.4	199 199 224	(207) 14.4 0.8	93 97 117	(102) 12.9 0.8	22 25 21	(23) 2.1 0.9	12 7 8	(9) 2.6 0.7
+	5000	47 SP 65 SP 67 SP	(60) 11.0 0.6	0 P 0 P 0 P	(0) 0.0 0.0	196 P 179 P 157 P	(177) 19.6 0.7	68 P 67 P 64 P	(66) 2.1 0.5	0 VP 0 VP 0 VP	(0) 0.0 0.0	10 SP 10 SP 8 SP	(9) 1.2 0.7
Positive controls	Name	2AA		2AA		2AA		2AA		BP		2AA
S9-Mix +	Concentration (μg/plate)	1		2		10		10		5		2
	No. colonies per plate	574 668 548	(597) 63.1 6.1	120 147 165	(144) 22.7 10.3	2106 2191 2232	(2176) 64.3 8.5	500 435 472	(469) 32.6 3.6	361 308 299	(323) 33.5 12.4	122 122 122	(122) 0.0 9.4

BP         Benzo(a)pyrene

2AA     2-Aminoanthracene

S           Sparse bacterial background lawn

V           Very weak bacterial background lawn

P           Precipitate

0.0        Fold increase over concurrent vehicle control

#        Standard deviation

 

 

Test Results: Experiment 2 – With Metabolic Activation & Pre-Incubation

Remark: due to constraints of this IUCLID field it was not possible to include this table. The complete "Any other information on results incl. tables of endpoint study record" can be found in attachment DCHK-NBA_Ames_add_info.pdf.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item was considered to be non-mutagenic under the conditions of this test.

This study has been initiated (study initiation date: 24 November 2010) for other purposes than to support a registration according to Regulation (EC) No 1907/2006 (REACH). It has not been carried out in compliance with the principles of good laboratory practice (GLP), which is, according to REACH Article 13 (4), required for toxicological tests. The study has been conducted according to the standard guideline(s). It was conducted in a facility operating to GLP within the UK national GLP monitoring programme and it was Quality Assurance audited to verify the integrity of the data and to ensure the report accurately reflects the data. The original data and the original final report are retained in the archives of the test facility. No formal claim of GLP compliance has been made for this study. The analytical characterisation of the test material has been performed according to GLP.
After assessing all information on this study, the registrant has concluded that the following conditions are met:
(1) adequacy for the purpose of classification and labelling and/or risk assessment;
(2) adequate and reliable coverage of the key parameters foreseen to be investigated in the corresponding test methods referred to in Article 13(3);
(3) exposure duration comparable to or longer than the corresponding test methods referred to in Article 13(3) if exposure duration is a relevant parameter; and
(4) adequate and reliable documentation of the study is provided.
Thus, according to REACH Annex XI, 1.1 ("Testing does not appear scientifically necessary - Use of existing data"), these existing data are considered to be acceptable for fulfilling the information requirements of this endpoint.

As a consequence, the overall reliability rating (according to Klimisch et al.) is considered to be between "reliable without restrictions" and "reliable with restrictions", but closer to "reliable without restrictions". Thus an overall Klimisch rating of 1 (reliable without restrictions) has been assigned.

Executive summary:

This study was designed to assess the mutagenic potential of test item using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames.

The bacterial strains S. typhimurium TA98, TA100, TA1535, TA1537, E. coli WP2uvrApKM101 and WP2pKM101 were treated with the test item at up to seven dose levels in triplicate. In experiment I, the plate incorporation method was followed and cultures with and without S9 mix were tested. In experiment II, the preincubation method was followed and only cultures with S9 mix were tested. The solvent, untreated and positive controls were also tested in triplicate.

The sensitivity of the assay and the efficacy of the S9-mix were validated by the results obtained for the solvent and positive controls.

In the first experiment (plate incorporation methodology) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of S9-mix at 5000 µg/plate. In the presence of S9-mix, weakened bacterial background lawns were noted for TA100, TA98 and TA1537 at 5000 µg/plate with substantial reductions in revertant colony frequency noted at the same dose level for TA1535, WP2uvrApKM101 and WP2pKM101. In the second experiment (pre-incubation methodology) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 1500 µg/plate (TA100) and at 5000 µg/plate (TA1535, TA98 and TA1537). There was no visible reduction in the viability of the bacterial background lawns of Escherichia coli strains WP2uvrApKM101 and WP2pKM101, however slight reductions in colony frequency were noted. The test item was tested up to the maximum recommended dose level (5000 µg/plate). A test item precipitate (fibrous in appearance) was noted at 5000 µg/plate in both experiments. This observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies, in excess of two‑fold greater than the concurrent solvent controls, were recorded for any of the bacterial strains, for any dose level of the test item, either with or without metabolic activation.

In conclusion, the test item was considered to be non-mutagenic under the conditions of this test.