(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-enecarboxylic acid

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:

2013 -11-04 till 2013-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2014

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:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Details on test material:

Identification: (-)-Shikimic acid
CAS No.: 138-59-0
Roche No.: Ro0642565-000
Batch: 1329R037/ Lot. No. 0700791872
Purity: Min. 98.0%
Expiry Date: 22 August 2014
Storage Conditions: At room temperature, protected from light*
Stability in Solvent: Not indicated by the Sponsor

* only valid for storage, not for test performance

No correction for purity was made.

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: soluble in DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

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 (strains TA 98, TA 100, and TA 102 or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, 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:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: unsoluble in water
- Precipitation: No precipitation observed
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
In experiment II, the number of colonies did not quite reach the lower limit of our historical control data in strain TA 100 in the solvent control with and without metabolic activation. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal back¬ground growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol¬ic activation.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table1     Summary of Experiment I

Study Name: 1584504	Study Code: Harlan CCR 1584504
Experiment: 1584504 VV Plate	Date Plated: 04/11/2013
Assay Conditions:	Date Counted: 07/11/2013

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	DMSO			20 ± 4	12 ± 2	24 ± 3	97 ± 8	498 ± 13
	Untreated			22 ± 2	10 ± 5	24 ± 5	102 ± 8	477 ± 1
	(-)-Shikimic acid	3 µg		18 ± 6	12 ± 3	23 ± 2	99 ± 11	541 ± 18
		10 µg		19 ± 4	12 ± 2	26 ± 6	97 ± 14	483 ± 17
		33 µg		20 ± 3	11 ± 1	24 ± 4	104 ± 10	526 ± 17
		100 µg		22 ± 3	8 ± 2	24 ± 4	88 ± 9	529 ± 16
		333 µg		24 ± 0	12 ± 5	24 ± 4	100 ± 22	420 ± 90
		1000 µg		24 ± 8	10 ± 3	26 ± 4	98 ± 4	498 ± 30
		2500 µg		20 ± 5	12 ± 2	24 ± 2	104 ± 10	497 ± 2
		5000 µg		13 ± 4	15 ± 4	30 ± 3	103 ± 3	476 ± 11
	NaN3	10 µg		2921 ± 77			2207 ± 29
	4-NOPD	10 µg				269 ± 46
	4-NOPD	50 µg			72 ± 12
	MMS	2.0 µL						5384 ± 568
With Activation	DMSO			28 ± 5	16 ± 4	34 ± 9	127 ± 13	590 ± 25
	Untreated			32 ± 1	21 ± 10	39 ± 4	141 ± 7	594 ± 14
	(-)-Shikimic acid	3 µg		28 ± 8	18 ± 1	29 ± 8	129 ± 8	605 ± 4
		10 µg		34 ± 7	14 ± 1	34 ± 1	133 ± 28	581 ± 45
		33 µg		25 ± 10	17 ± 3	37 ± 1	142 ± 12	578 ± 17
		100 µg		32 ± 8	18 ± 2	32 ± 4	127 ± 3	629 ± 29
		333 µg		29 ± 1	19 ± 4	38 ± 3	124 ± 3	544 ± 33
		1000 µg		31 ± 4	21 ± 4	41 ± 6	120 ± 8	531 ± 35
		2500 µg		24 ± 2	16 ± 6	38 ± 4	119 ± 4	640 ± 98
		5000 µg		33 ± 6	14 ± 6	33 ± 0	112 ± 6	605 ± 15
	2-AA	2.5 µg		536 ± 40	340 ± 21	4086 ± 165	4271 ± 168
	2-AA	10.0 µg						5347 ± 502

 

Key to Positive Controls
NaN3 2-AA MMS 4-NOPD	sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine

 

 

Table2     Summary of Experiment II

Study Name: 1584504	Study Code: Harlan CCR 1584504
Experiment: 1584504 HV2 Pre	Date Plated: 28/11/2013
Assay Conditions:	Date Counted: 03/12/2013

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	DMSO			21 ± 6	9 ± 3	23 ± 11	75 ± 4	381 ± 12
	Untreated			25 ± 10	9 ± 5	24 ± 6	94 ± 10	479 ± 39
	(-)-Shikimic acid	33 µg		24 ± 2	7 ± 2	20 ± 3	82 ± 12	425 ± 52
		100 µg		24 ± 8	9 ± 4	21 ± 2	75 ± 8	376 ± 19
		333 µg		28 ± 3	7 ± 3	21 ± 4	88 ± 10	398 ± 20
		1000 µg		24 ± 4	7 ± 2	25 ± 8	85 ± 7	376 ± 31
		2500 µg		21 ± 2	10 ± 4	24 ± 5	71 ± 4	334 ± 43
		5000 µg		22 ± 8	8 ± 5	26 ± 2	85 ± 13	381 ± 54
	NaN3	10 µg		2744 ± 64			2045 ± 197
	4-NOPD	10 µg				411 ± 9
	4-NOPD	50 µg			83 ± 2
	MMS	2.0 µL						3599 ± 221
With Activation	DMSO			17 ± 4	22 ± 8	41 ± 8	84 ± 11	438 ± 15
	Untreated			18 ± 4	18 ± 4	32 ± 9	94 ± 3	418 ± 11
	(-)-Shikimic acid	33 µg		19 ± 8	17 ± 5	38 ± 9	88 ± 8	395 ± 9
		100 µg		18 ± 8	20 ± 5	33 ± 9	75 ± 5	441 ± 29
		333 µg		18 ± 4	14 ± 6	41 ± 6	94 ± 17	411 ± 74
		1000 µg		14 ± 5	17 ± 4	29 ± 6	92 ± 6	470 ± 15
		2500 µg		22 ± 4	20 ± 9	30 ± 3	88 ± 10	451 ± 62
		5000 µg		19 ± 4	17 ± 6	41 ± 3	96 ± 13	388 ± 20
	2-AA	2.5 µg		510 ± 23	224 ± 18	2401 ± 292	1969 ± 90
	2-AA	10.0 µg						986 ± 70

 

Key to Positive Controls
NaN3 2-AA MMS 4-NOPD	sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine

 

Applicant's summary and conclusion

Conclusions:

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

In conclusion, it can be stated that during the described mutage¬nicity test and under the experimental conditions reported, the (-)-Shikimic acid did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study was performed to investigate the potential of (-)-Shikimic acid to induce gene muta­tions according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                33; 100; 333; 1000; 2500; and 5000 µg/plate

 

No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol­ic activation.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with (-)-Shikimic acid at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.