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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
AMES test
Type of information:
experimental study
Adequacy of study:
key study
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)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
Adipic acid, ammonium salt
EC Number:
242-809-3
EC Name:
Adipic acid, ammonium salt
Cas Number:
19090-60-9
Molecular formula:
C6H10O4.xH3N
IUPAC Name:
ammonium 5-carboxypentanoate
Test material form:
solid: crystalline

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:
The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH, Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
Test concentrations with justification for top dose:
The Ammonium Adipate concentrations investigated in the Initial and Confirmatory
Mutation Tests: ±S9: 5000, 1600, 500, 160, 50 and 16 µg/plate;

Recommended maximum concentration according to the OECD 471 guidance is 5000 µg/plate.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine, NPD, Sodium azide, SAZ, 9-Aminoacridine, 9AA, Methyl methanesulfonate, MMS
Details on test system and experimental conditions:
Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia
coli WP2 uvrA were obtained from:
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures. The identification codes, arrival
date and expiry dates of the actual applied stock cultures are summarized in Table 3.
Rationale for test conditions:
Based on the results of the preliminary tests, a stock solution with a concentration of
50 mg/mL was prepared from the test item with ultrapure water, which was diluted by serial
dilutions to obtain six dosing solutions for lower doses. The maximum test concentration
was 5000 µg/plate (±S9). At the concentration choice the guideline criterion for soluble,
non-toxic test compounds was taken into consideration based on the recommendations in
OECD 471 guideline [6], where the recommended maximum test concentration is
5000 µg/plate. Therefore, in this test the highest test item concentration was 5000 µg/plate
(±S9). As indicated in section 6.1.2, this top concentration was a non-precipitating
concentration
Evaluation criteria:
The colony numbers on the controls (untreated, vehicle, positive) and the test item plates
were determined (counted manually, evaluated by unaided eye), the mean values and
appropriate standard deviations and mutation rates were calculated.
* : untreated, vehicle or positive control
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.

Results and discussion

Test resultsopen allclose all
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
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
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:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
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:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
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:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
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:
valid
True negative controls validity:
not specified
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The reported data of this mutagenicity assay show that under the experimental conditions
applied, the test item did not induce gene mutations by base pair changes or frameshifts
in the genome of the investigated strains: Salmonella typhimurium TA98, TA100,
TA1535 and TA1537 and of Escherichia coli WP2 uvrA.
In conclusion, the test item Ammonium Adipate has no mutagenic activity on the
applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test itemAmmonium Adipate was tested with regard to a potential mutagenic activity
using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella
typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the
tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in
the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of
Phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Solubility Test, a Preliminary Concentration Range
Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation
Test), and a Confirmatory Mutation Test (Pre-Incubation Test).
Based on the results of the Solubility Test and the Concentration Range Finding Test the test
item was dissolved in ultrapure water (ASTM Type I).
At the concentration choice the cytotoxicity and the solubility of the test item (obtained in
the Concentration Range Finding Test) were taken into consideration and based on the
recommendations of OECD 471 guideline [6] the following concentrations of the test item
were prepared and investigated in the Initial and Confirmatory Mutation Tests:
±S9: 5000, 1600, 500, 160, 50 and 16 µg/plate;
No precipitation of the test item was observed on the plates in the examined bacterial strains
at any examined concentration level (±S9) throughout the study.
The test item did not show inhibitory, cytotoxic effects in the performed experiments. The
colony and background lawn development were not affected in any case; the obtained
revertant colony number decreases (compared to the revertant colony numbers of the vehicle
control) remained within the biological variability range of the applied test system.
The revertant colony numbers of vehicle control (ultrapure water) plates with and without
S9 demonstrated the characteristic mean number of spontaneous revertants that was in line
with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological
relevant increases (more than 3-fold increase) in induced revertant colonies and the number
of revertants fell in the corresponding historical control ranges, thereby meeting the criteria
for the positive control in all experimental phases, in all tester strains.
No biologically relevant increases in revertant colony numbers were observed in revertant
colony numbers of any of the five test strains following treatment withAmmonium
Adipateat any concentration level, either in the presence or absence of metabolic activation
(S9 Mix) in the performed experiments.
The reported data of this mutagenicity assay show that under the experimental conditions
applied, the test itemdid not induce gene mutations in the genome of the strains of
Salmonella typhimuriumTA98, TA100, TA1535 and TA1537 and ofEscherichia coli
WP2uvrA.
In conclusion, the test itemAmmonium Adipate has no mutagenic activity on the
applied bacterium tester strains under the test conditions used in this study.