3-tert-butyladipic acid

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:

2017-08-22 to 2017-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

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:

3-tert-butyladipic acid

EC Number:

233-759-3

EC Name:

3-tert-butyladipic acid

Cas Number:

10347-88-3

Molecular formula:

C10H18O4

IUPAC Name:

3-tert-butylhexanedioic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Changzhou Sunlight Pharmaceutical Co., Ltd., China; 20170601
- Expiration date of the batch: June 2018
- Purity test date: 99.72 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions.

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver)

Test concentrations with justification for top dose:

5, 16, 50, 160, 500, 1600, 5000 µg/plate ± S9 mix

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The pre-tests resulted in a good solubility of the test substance.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

NPD

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine

Remarks:

without S9 mix (4 μg/plate, TA 98)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

SAZ

Positive control substance:

sodium azide

Remarks:

Without S9 mix (2 µg/plate; TA100, TA1535)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

9AA

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix (50 µg/plate; TA 1537)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

MMS

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9 mix (2 µL/plate, E. coli)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2AA

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix (2 µg/plate, all Salmonella strains; 50 µg/plate, E.coli)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 hours at 37 °C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results. A statistical evaluation of the results was not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

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

Untreated negative controls validity:

not examined

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

Untreated negative controls validity:

not examined

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:

not valid

Untreated negative controls validity:

not examined

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No
- Water solubility: No
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
The revertant colony numbers of vehicle control plates in both strains with and without S9 mix in the pre test were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
In the Informatory Toxicity Test, inhibitory effects of the test item were not observed. The colony and background lawn development were not affected in any case. All of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).

Any other information on results incl. tables

Table Results

Experiment 1 (SPT)
Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E. coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results with S9
Untreated control	11.7	7.7	23	99.7	29
DMSO control	12	7.7	23.7	91.3	34.7
Ultrapure Water Control	-	-	-	-	-
5000	18.7	6	32.3	107	42
1600	14.3	6.3	31.3	99	32.7
500	11	7.3	24.7	112	29.3
160	11.7	8	28.7	111.7	32.3
50	10	6.3	24.3	107.7	42
16	13.7	8.3	25	116.3	27.7
Positive control	187.3	188.7	1217.3	2053.3	220.7
	Results without S9
Untreated control	13	6.7	27.3	102.3	32
DMSO control	13.3	8.3	21.3	93.3	25
Ultrapure Water Control	12.7	-	-	92.7	29.3
5000	12.3	9.7	21.3	101.3	28
1600	12.3	8.3	19.7	97.7	30.3
500	13.7	9	19.3	94.7	32
160	13.7	8.7	22	94	32.7
50	11.7	7.3	18	92.7	33.3
16	10.3	7	22	93.9	26.3
Positive control	901.3	607.3	332	1172.3	773.3
Experiment 2 (PIT)
Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E. coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results with S9
Untreated control	12	7.3	27.7	99.7	43
DMSO control	14.7	7.7	21	100.3	42.3
Ultrapure Water Control	-	-	-	-	-
5000	12.7	6	23	105.3	34.7
1600	14.7	6	20.7	106.7	41
500	13	9.3	19.3	98	33.3
160	10.3	7.3	25	110	30
50	13.3	7.3	24	108	29.3
16	13	6.7	20.3	110	26.7
Positive control	232.7	182	1672	2933.3	164.3
	Results without S9
Untreated control	10.7	9	20.7	94	37.3
DMSO control	12	8.3	20	92.3	32.3
Ultrapure Water Control	13.3	-	-	93.7	41.7
5000	10.7	8.3	27	91.7	43
1600	10.7	8	26.3	82.7	44.7
500	12.7	8.7	24.3	84.3	48.7
160	10.7	9.3	20.7	83.3	56.3
50	11	7.7	17	80.3	48
16	11.3	10	20	77.7	54
Positive control	2072	574.7	294.7	1912	1757.3

Applicant's summary and conclusion

Conclusions:

Under the conditions of the above assay, the test substance tested formulated in a suitable vehicle (DMSO) did not show any mutagenic potential.

Executive summary:

In a reverse mutation assay in bacteria according to OECD guideline 471, strains TA98, TA1537, TA1535 and TA100of S. typhimuriumand Escherichia coli WP2 uvrA were investigated. The test item was dissolved in dimethyl sulfoxide (DMSO) and concentrations of 5000, 1600, 500; 160; 50 and 16 μg/plate were examined.

Five bacterial strains were used to investigate the mutagenic potential of the test substance in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.

In the performed experiments all of the validity criteria regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled.

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix).

Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments. They were mostly within the actual historical control data ranges. Furthermore, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

In the performed experiments an inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test substance is considered non-mutagenic in this bacterial reverse mutation assay.