Hexanoic acid, 6-[(3,5,5-trimethyl-1-oxohexyl)amino]-, compd. with 2,2',2''-nitrilotris[ethanol] (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 5 JUL 1988 to 25 JUL 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was performed according to guideline OECD 471

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

It is hypothesized that the target chemical and the following chemicals as source chemicals should exhibit comparable toxicity profiles:
• 6-(isononanoylamino)hexanoic acid
• 3,5,5-trimethylhexanoic acid

It is proposed to use the toxicity data of the mentioned source chemicals to fulfill the data requirement for the target chemical on the human health endpoint 'Genotoxicity'. The first chemical is the corresponding acid of the target chemical. 3,5,5-trimethylhexanoic acid is a presumed metabolite.

The underlying scientific rationale for the use of corresponding acid as source chemical is apparent. The target chemical is the 2,2`,2``-nitrilotriethanol (TEA) salt of the source chemical. The source chemical is a weak acid due to the terminal carboxylic acid moiety and can be neutralized/dissolved in aqueous system by reaction with base such as TEA. The target chemical can be formally described as carboxylate of the source chemical. As the carboxylate and carboxylic acid are inter-convertible, it is apparent that source and target chemicals are inter-convertible and should exhibit comparable toxicity profile. The base 2,2`,2``-nitrilotriethanol is a well-investigated substance and is considered to be less relevant for the toxicological assessment.
The underlying scientific rationale for the use of 3,5,5-trimethylhexanoic acid as source chemical is based on the metabolism consideration. Upon absorption, the target chemical is expected to undergo a degradation process, resulting in the systemic release of 3,5,5-trimethylhexanoic acid, thereby providing the justification for the read-across especially for the mid- and long term toxicities such as repeated dose toxicity and reproduction toxicity.

The proposed approach applies for all exposure routes (oral/dermal/inhalation), because both the target chemical and source chemicals are expected to be bioavailable by all exposure routes and the systemic release of the presumed metabolite is less dependent on exposure route.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

Target chemical:
6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol; CAS: 85702-79-0

Source chemicals:
6-(Isononanoylamino)hexanoic acid; CAS: 71902-23-3
3,5,5´-trimethylhexanoic acid; CAS: 3302-10-1

The target chemical is a mono-substituent substance, the analytical purity being >99%. The source chemicals are the raw materials (3,5,5´-trimethylhexanoic acid and 6-(isononanoylamino) hexanoic acid) of the target chemical. A toxicity difference due to different impurity profiles is not likely to occur.

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

Justification for the use of 6-(isononanoylamino)hexanoic acid as source chemical:

- The target chemical is an ionic compound that results from the neutralization reaction of the given source chemical and 2,2`,2``-nitrilotriethanol. When it is dissolved in an aqueous system or in a biological fluid, an immediate dissociation occurs to give the source chemical and the base, thereby explaining the expected comparable toxicity profile to that of target chemical.
A significant toxicity contribution of 2,2`,2``-nitrilotriethanol is not expected. 2,2`,2``-nitrilotriethanol is a well investigated substance. It is of low toxicity and the available kinetic data are demonstrative of efficient elimination mechanisms in animal models.

- In order to verify the expected toxicity comparability, the given source and the target chemicals were investigated under identical testing conditions. Both substances exhibited comparable findings after 7-day oral application to rat:
liver and kidney enlargement
decrease of eosinophil counts
peroxisome proliferation in the liver
The decrease of eosinophil counts is possibly a transient effect, associated with the peroxisome proliferation stimulating effect of test compounds. No such findings were present after 28-day treatment of the target chemical.

- Comparable findings were obtained in the 28-day oral toxicity study for the target chemical and in the above mentioned two 7-day repeated oral toxicity studies with the source and the target substance. Further special histopathological investigation revealed the alpha-2u-globulin accumulation in male kidneys and the peroxisome proliferation in liver.

Justification for the use of 3,5,5-trimethylhexanoic acid as source chemical:

- 3,5,5-trimethylhexanoic acid is the presumed metabolite of 6-(isononanoylamino)hexanoic acid. It is also the presumed metabolite of 6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol.
This view is based on the results of the metabolites investigation in degradation samples obtained in a Zahn-Wellens test. The β-oxidation at the terminal carboxylic acid moiety in combination with hydrolysis at amide bond could be assumed as the degradation pathway leading to 3,5,5-trimethylhexanoic acid as a stable metabolite.
Also the hydrolysis at the amide moiety is thinkable. The hydrolysis products would be then 3,5,5-trimethylhexanoic acid and 6-aminocaproic acid. The latter compound is a drug known as amicar and is expected to be far more rapidly eliminated than 3,5,5-trimethylhexanoic acid. A significant toxicity attribution of 6-aminocaproic acid cannot be derived.
One literature article was found, describing further biotransformation of 3,5,5-trimethylhexanoic acid in rat: gamma-lactone of 3,5,5-trimethylhexanoic acid is formed, which may be the ultimate toxicant for the alpha-2u-globulin accumulation in kidneys.
As mentioned above no significant toxicity contribution of 2,2`,2``-nitrilotriethanol is expected.

-The findings in the 28-day oral toxicity study on the 3,5,5-trimethylhexanoic acid are comparable to those found in the repeated dose toxicity studies of the target chemical and 6-(isononanoylamino)hexanoic acid:
- liver and kidney enlargement
- peroxisome proliferation in the liver
- The alpha-2u-globulin accumulation in kidneys.

4. DATA MATRIX

Data matrix and other information see the attached read-across justification in chapter 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Test guideline 5

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,5,5-trimethylhexanoic acid

EC Number:

221-975-0

EC Name:

3,5,5-trimethylhexanoic acid

Cas Number:

3302-10-1

Molecular formula:

C9H18O2

IUPAC Name:

3,5,5-trimethylhexanoic acid

Details on test material:

Name of test material (as cited in study report): Isononansäure, 3,5,5-trimethylhexanoic acid, Batch- No. IVNE 015/ IVNS, no further details on composition or purity

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of livers from Sprague-Dawley rats, induced with Aroclor 1254

Test concentrations with justification for top dose:

first experiment: 4, 20, 100, 500, 2500, 10000 µg/plate
second experiment: 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: sodium azide, 9-aminoacridine, 2-nitrofluorene; with S9: 2-aminoanthracene, benzo[a]pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: 2 ml of molten top agar (plate incorporation) with 100 µl of overnight nutrient broth culture of bacteria tester strain and 100 µl of test compound solution with or without 500 µl of S9 mix are poured into a petri dish with a layer of minimal agar. His+ revertants were counted after 48-72 h incubation at 37 °C in the dark.

Evaluation criteria:

A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn (controlled microscopically) were used as indicator for toxicity.

Statistics:

Statistical analysis was not stated.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 µg/plate and above in the most strains

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test substance was not mutagenic with or without metabolic activation.

Executive summary:

The test substance was not mutagenic in an Ames-test with the Salmonella typh. strains TA98, TA100, TA1535, TA1537 and TA1538 and E.coli WP2uvrA in concentrations up to 10000 µg/plate, both with or without metabolic activation (Hoechst AG, 1988).