Octanoic acid, compound with dicyclohexylamine (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Octanoic acid:

Octanoic acid is a linear saturated fatty acid and as well as other similar fatty acids is ubiquitous in nature: Octanoic acid is naturally present in many types of food in its free form or as triglyceride.

As a normal dietary constituent octanoic acid is devoid of any genotoxic property.

DCHA:

Dicyclohexylamine showed no mutagenic activity in standard Ames test with and without metabolic activation system but induced chromosomal aberrations in Chinese hamster lung (CHL) cells at high concentrations and short exposure time (exposure time: 6 hours (a) with S9-mix.at 600 μg/ml; (b) without S9-mix: at 800 and 1000 μg/ml).

Ames Test:

Dicyclohexylamin did not induce gen mutations in the bacterial reverse mutation assay in the strains Salmonella typhimurium TA98, TA100, TA1535, TA1537, Escherichia coli WP2uvrA/pKM101 with and without metabolic activation.

Chromosome aberration:

With the 6-hr short-term treatment, structural chromosomal aberrations including gaps were induced at 600 µg/mL(10.0 %) without an S9 mix and at 800 and 1000 µg/mL(13.5 and 31.0 %) with an S9 mix. Polyploidy was not induced in any treatment group.

Cytotoxicity was observed at 400 and 500 µg/mL after 48-hr continuous treatment without an S9 mix, and at 600 and 800 µg/mL after 6-hr short-term teatment without an S9 mix.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Details about test parameter and details of method are not reported.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 activation

Test concentrations with justification for top dose:

No details are reported.

Vehicle / solvent:

No details are reported.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

No details are reported.

Evaluation criteria:

No details are reported.

Statistics:

No details are reported.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Octanoic acid was inactive in Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100.

Executive summary:

In the bacterial reverse mutation assay octanoic acid was inactive in Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

According to REACH Annex XI, section 1, the standard testing regime may be adapted if testing does not appear scientifically necessary.
In aqueous systems Octanoic acid, compound with N-cyclohexylcyclohexanamine (1:1) will rapidly dissociate to octanoic acid and dicyclohexylamine.
Octanoic acid is a linear saturated fatty acid and is ubiquitous in nature. According to Efsa, (European Food Safety Authorisation) caprylate (octanoic acid) is without any structural alert for genotoxicity (citation):
“Caprylic acid is a natural component of the diet usually esterified in the form of triacylglycerol; it is found in the fat content of dairy products such as milk (0.3 %) and butter (0.8 %), and also in palm kernel oil (3-4 %) and coconut oil (9-10 %).
The average dietary exposure to caprylate is considered to be in the range of 330 to 410 mg/day, and at high percentile dietary exposure up to 992 mg/day. The Panel noted that caprylate (octanoic acid) is a normal dietary constituent, absorbed from the intestine to the portal circulation, rapidly metabolised into carbon dioxide and two-carbon fragments and is without any structural alert for genotoxicity.”
(Efsa, (European Food Safety Authorisation): “SCIENTIFIC OPINION, Calcium caprylate and magnesium caprylate added for nutritional purposes as sources of calcium and magnesium to food supplements”, The EFSA Journal, 1146, 1-20 (2009))

Also according to Umweltbundesamt GmbH on behalf of Federal Ministry of Agriculture, Forestry, Environment and Water Management octanoic acid is not genotoxic (citation):
“Detailed knowledge is available on the metabolic pathways that are similar for all fatty acids: complete catabolism for energy supply or conversion to fat suitable for storage.
Decanoic acid and Octanoic acid are linear saturated fatty acids and they as well as other similar fatty acids are ubiquitous in nature: Decanoic acid and Octanoic acid are naturally present in many types of food in its free form or as triglyceride. Uptake as natural food source from cheese or coconut oil may be estimated to be significantly above 20 mg/ person day.”
“Considering all the negative genotoxicity results for Octanoic acid and for Decanoic acid and considering the absence of structural alerts of the active substances and the known impurities as well as all arguments listed in chapter 4.7 (bullet points) the overall conclusion is that neither Decanoic acid nor Octanoic acid are genotoxic.”
(Umweltbundesamt GmbH on behalf of Federal Ministry of Agriculture, Forestry, Environment and Water Management: “CLH report, Proposal for Harmonised Classification and Labelling, Based on Regulation (EC) No 1272/2008 (CLP Regulation), Annex VI, Part 2, Substance Name: Octanoic acid”, Version number: 3, 2012-06-04; available under: http://echa.europa.eu/documents/10162/a7fbd63c-a2eb-480e-aad7-a28eee509507)

Therefore testing does not appear scientifically necessary as any additional testing would not contribute any additional knowledge to the already existing comprehensive toxicological database for octanoic acid.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: literature data

Adequacy of study:

weight of evidence

Study period:

2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

other: review of toxicological properties of medium chain triglycerides

Species / strain / cell type:

Saccharomyces cerevisiae

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

other: TA 1535, TA 1537, TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

no information available

Vehicle / solvent:

no information available

Details on test system and experimental conditions:

no information available

Rationale for test conditions:

no information available

Evaluation criteria:

no information available

Statistics:

no information available

Key result

Species / strain:

other: TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

Saccharomyces cerevisiae

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

The results of the mutagenicity tests in Salmonella typhimurium TA 1535, TA 1537, TA 1538 and Saccharomyces cerevisiae strains with caprylic acid indicate that the substance does not have the potential to be mutagenic.

Executive summary:

The results of the mutagenicity tests in Salmonella typhimurium TA 1535, TA 1537, TA 1538 and Saccharomyces cerevisiae strains with caprylic acid indicate that the substance does not have the potential to be mutagenic.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Remarks:

read-across from hydrolysis product

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In aqueous systems Octanoic acid, compound with dicyclohexylamine (1:1) is hydrolytically very unstable so that in aqueous solution a rapid decomposition to the educts can be observed.The substance will rapidly dissociate to octanoic acid and dicyclohexylamine. Therefore Octanoic acid, compound with dicyclohexylamine (1:1) is not stable at “standard” testing conditions representative for human and environmental exposure. Hence, It is fully justified to apply the read-across methodology by use of the respective data from the breakdown/decomposition products to describe the (eco)toxicological behaviour of the substance to be registered.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: octanoic acid, dicyclohexylamine
Target: Octanoic acid, compound with dicyclohexylamine (1:1)


3. ANALOGUE APPROACH JUSTIFICATION
The corresponding breakdown products of Octanoic acid, compound with dicyclohexylamine (1:1) were identified by Ultra Performance Liquid Chromatography - tandem Mass Spectrometer (UPLC-MS) as Octanoic acid and Dicyclohexylamine.
Further evaluation of the hydrolysis time demonstrated that in aqueous solution the decomposition time is smaller than 8 minutes.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

In the bacterial reverse mutation assay octanoic acid was inactive in Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Remarks:

read-across from hydrolysis product

Adequacy of study:

weight of evidence

Study period:

2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In aqueous systems Octanoic acid, compound with dicyclohexylamine (1:1) is hydrolytically very unstable so that in aqueous solution a rapid decomposition to the educts can be observed.The substance will rapidly dissociate to octanoic acid and dicyclohexylamine. Therefore Octanoic acid, compound with dicyclohexylamine (1:1) is not stable at “standard” testing conditions representative for human and environmental exposure. Hence, It is fully justified to apply the read-across methodology by use of the respective data from the breakdown/decomposition products to describe the (eco)toxicological behaviour of the substance to be registered.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: octanoic acid, dicyclohexylamine
Target: Octanoic acid, compound with dicyclohexylamine (1:1)


3. ANALOGUE APPROACH JUSTIFICATION
The corresponding breakdown products of Octanoic acid, compound with dicyclohexylamine (1:1) were identified by Ultra Performance Liquid Chromatography - tandem Mass Spectrometer (UPLC-MS) as Octanoic acid and Dicyclohexylamine.
Further evaluation of the hydrolysis time demonstrated that in aqueous solution the decomposition time is smaller than 8 minutes.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: TA 1535, TA 1537, TA 1538

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Saccharomyces cerevisiae

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

no information available

Vehicle / solvent:

no information available

Details on test system and experimental conditions:

no information available

Rationale for test conditions:

no information available

Evaluation criteria:

no information available

Statistics:

no information available

Key result

Species / strain:

other: TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

Saccharomyces cerevisiae

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium TA98, TA100, TA1535, TA1537, Escherichia coli WP2uvrA

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

DRF: -S9 mix; 156, 313, 625,1250, 2500, 5000//g/plate
+S9 mix; 156, 313, 625,1250, 2500,5000 µg/plate
Reverse Mutation Test, Main Study:
-S9 mix; 78,156, 313, 625,1250,2500 µg/plate
+S9 mix; 78,156, 313, 625,1250, 2500 /µg/plate
Reverse Mutation Test, Confirmation:
+S9 mix; 750, 1000,1250,1500,1750,2000,2250,2500 µ/g/ plate

Vehicle / solvent:

Acetone

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2- (2-Furyl) -3- (5-nitro-2-furyl) acrylamide, 2-Aminoanthracene

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

above 2500 µg/plate

Vehicle controls validity:

not specified

Untreated 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:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated 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:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Dicyclohexylamine did not induce mutations in the S. typhimurium and E. coli strains. Toxicity was observed at more than 2500 /µg/plate(all strains) with and without an S9 mix. In the confirmative test, toxicity was observed at more than 1500 /µg/plate(TA100)with S9 mix.

Executive summary:

Dicyclohexylamine did not induce mutations in the S. typhimurium and E. coli strains. Toxicity was observed at more than 2500 /µg/plate(all strains) with and without an S9 mix. In the confirmative test, toxicity was observed at more than 1500 /µg/plate(TA100)with  S9 mix.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: Ames, Mutat. Res. 31,347(1975); Maron, Mutat. Res.113,173(1983)

Deviations:

not specified

Principles of method if other than guideline:

Ames, Mutat. Res. 31,347(1975); Maron, Mutat. Res.113,173(1983)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium TA98, TA100, TA1535, TA1537, Escherichia coli WP2uvrA/pKM101

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

(1) 0, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 μg/plate in DMSO (2) 0, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/plate in DMSO

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide 2-Aminoanthracene

Evaluation criteria:

Twofold rule was used for data evaluation. the chemicals are considered to be mutagenic when dose-related increase in revertant colony count is observed and the number of revertant colonies per plate with the test substance is more than twice that of the negative control (solvent control) and when a reproducibility of test result is observed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Dicyclohexylamin did not induce gen mutations in the bacterial reverse mutation assay in the strains Salmonella typhimurium TA98, TA100, TA1535, TA1537, Escherichia coli WP2uvrA/pKM101 with and without metabolic activation.

Executive summary:

Dicyclohexylamin did not induce gen mutations in the bacterial reverse mutation assay in the strains Salmonella typhimurium TA98, TA100, TA1535, TA1537, Escherichia coli WP2uvrA/pKM101 with and without metabolic activation.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

other: in vitro chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung (CHL) cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9: Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9 mix (24 and 48-hr continuous treatment): 0, 100, 200, 250, 300,400, 500 jug/mL
-S9 mix (6-hr short-term treatment): 0,100, 200,400, 600, 800,1000 fug/mL
+S9 mix (6-hr short-term treatment): 0,100, 200,400, 600, 800,1000 ¡ug/mL

Vehicle / solvent:

Acetone

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: N-methyl-N'-nitro-N-nitrosoguanidine

Species / strain:

other: Chinese hamster lung (CHL) cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

lowest 0,6 mg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

With the 6-hr short-term treatment, structural chromosomal aberrations including gaps were induced at 600 µg/mL(10.0 %) without an S9 mix and at 800 and 1000 µg/mL(13.5 and 31.0 %) with an S9 mix. Polyploidy was not induced in any treatment group.
Cytotoxicity was observed at 400 and 500 µg/mL after 48-hr continuous treatment without an S9 mix, and at 600 and 800 µg/mL after 6-hr short-term teatment without an S9 mix.

Executive summary:

With the 6-hr short-term treatment, structural chromosomal aberrations including gaps were induced at 600 µg/mL(10.0 %) without an S9 mix and at 800 and 1000 µg/mL(13.5 and 31.0 %) with an S9 mix. Polyploidy was not induced in any treatment group.

Cytotoxicity was observed at 400 and 500 µg/mL after 48-hr continuous treatment without an S9 mix, and at 600 and 800 µg/mL after 6-hr short-term teatment without an S9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

DCHA:

In vivo Erythrocytes Micronucleus Test:

The test substance Dicyclohexylamine, was tested for the assessment of cytogenetic damage using micronucleus test.

The count of polychromatic erythrocytes with micronuclei after 24-hour and 48-hour exposition was raised with increasing dose level in both males and females. Statistically significant increase in the count of micronucleated immature erythrocytes compared to the control was recorded at the highest dose level of 200 mg/kg and middle dose level 100 mg/kg.

Under the given test conditions, the test substance, Dicyclohexylamine, has positive result in micronucleus test.

In vivo Spermatogonial Chromosome Aberration test:

Dicyclohexylamine tested up to the maximum tolerated dose of 250 mg Dicyclohexylamine/kg b.w. by oral administration showed no mutagenic properties in the mammalian spermatogonial chromosome aberration test of the rat at the two tested sampling times of 24 hours and 48 hours. In the same system, Mitomycin C (positive reference item) induced significant damage.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.11.2013 - 14.2.2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian germ cell cytogenetic assay

Specific details on test material used for the study:

Designation: Dicyclohexylamine
Chemical name: Dodecahydrodiphenylamine
CAS no.: 101-83-7
EC no.: 202-980-7
Batch no.: 89030400
Characteristics: Colourless liquid, strong fishy odour
Content:
99.62% Diclohexamine
0.330% Organic impurities
< 0.050% Water

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: 267 - 335 g
- Fasting period before study: approx. 16 hours before administration
- Housing:groups of 2 - 3 in MAKROLON cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: at least 5 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +- 3°C
- Humidity (%): at least 30% but not exceeding 70%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Sesame oil

Details on exposure:

Administration volume: 20 mL/kg b.w.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

single dose

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Positive control(s):

Designation: Mitomycin C4
Dose level: 5 mg/kg b.w.
Route of administration: Intraperitoneal injection
Vehicle: Aqua ad iniectabilia
Administration volume: 20 mL/kg b.w.

Tissues and cell types examined:

spermatogonial cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose levels should cover a range from the maximum toxicity to little or none.The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. The highest dose may also be defined as a dose that produces some indication of toxicity in the spermatogonial cells (e.g. reduction in the ratio of spermatogonial mitoses to first and second meiotic metaphases; this reduction should not exceed 50%).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Three dose levels are used for the first sampling time (24 h).Only the highest dose is used at the later sampling time (48 h).

DETAILS OF SLIDE PREPARATION:
Seven (7) hours prior to the sampling time, the animals received 4 mg Colchicine6/kg b.w. i.p.. The animals were sacrificed by ether. After having removed the tunica albuginea, the seminiferous tubules of both testicles were exposed to hypotonic 1% sodium citrate solution for 20 minutes. Afterwards, still in toto, the seminiferous tubules were fixed in freshly prepared methanol/glacial acetic acid (3 + 1) fixative and the samples were left overnight (about 16 hours) at low temperature (0°C - 4°C). In order to prepare the slides the samples were centrifuged and the fixative was completely removed with a Pasteur pipette. The samples were gently resuspended by adding 60% acetic acid. Approx. 50 μL of the cell suspension were dropped onto a slide prewarmed to 48C and spread. The air-dried cells were stained in 10% Giemsa (in buffered phosphate solution, pH 7.2) for 45 minutes. Afterwards the slides were mounted. At least two slides were prepared per animal.

METHOD OF ANALYSIS:
Slides for evaluation were coded before microscopic analysis and examined at a magnification of 1000 x (Planapochromat 100/1.25). The mitotic index was determined by counting the number of metaphases per 1000 cells in each cell preparation. The mean mitotic index of the animals per group was compared with the mean mitotic index of the negative control (mitotic index: 1.0).
The analysis of structural aberrations (chromosome- and chromatid type) was carried out in 200 cells per animal. Cells with an incomplete number of centromeres or insufficient spreading were not used for analysis.

Evaluation criteria:

Mitotic index
% of cells with gaps
% of cells with aberrations including gaps
% of cells with aberrations excluding gaps

Statistics:

The assessment was carried out by a comparison of the samples with the positive and the vehicle references, using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity data).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Dose Range Finding Study:

Three dose levels of 125, 250 and 500 mg Dicyclohexylamine/kg b.w. were tested. A dose level of 125 mg/kg b.w. revealed slight signs of toxicity 60 minutes after administration. 250 mg Dicyclohexylamine/kg b.w. caused slight to moderate signs of toxicity 15 minutes to 3 hours after administration. The high dose level of 500 mg/kg b.w. revealed slight to severe signs of toxicity 15 to 60 minutes and death within 3 hours after administration.

Main Study:

For the main study three ascending doses of 62.5, 125 or 250 mg Dicyclohexylamine/kg b.w., p.o. were administered. Further groups received the vehicle (sesame oil) and one further group the positive reference item Mitomycin C (5 mg/kg b.w., i.p.). Each group consisted of 7 male rats.

No signs of systemic toxicity were noted at 62.5 mg/kg b.w.. A dose level of 125 mg/kg b.w. revealed slightly reduced motility, slight ataxia and slightly reduced muscle tone 60 minutes after administration. The high dose level of 250 mg/kg b.w. revealed slightly to moderately reduced motility, slight to moderate ataxia, slightly to moderately reduced muscle tone and slight to moderate dyspnoea 15 minutes to 6 hours as well as slight to severe tonic convulsions 30 minutes to 6 hours and slight pilo-erection 30 minutes to 3 hours after administration (sacrifice after 24 or 48 hours).

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Dicyclohexylamine ranged from 0.8% to 1.9%. These results were within the normal range as no significant difference was observed compared to negative control (0.7% or 1.1%). These results were within the range of published historical negative control data8 (fragments: 1.2±0.44%, exchanges: 0.8±0.44%, damaged chromosomes: 0.8±0.44%).

The percentage of cells with gaps was also within the range of the negative control (treated groups: 2.1% to 3.3%; control group: 1.8% to 3.9%).

No reduction of the mean mitotic index of the animals per group compared with the mean mitotic index of the negative control was noted. The positive control, Mitomycin C, induced significant levels of chromosomal aberrations.

Conclusions:

Dicyclohexylamine tested up to the maximum tolerated dose of 250 mg Dicyclohexylamine/kg b.w. by oral administration showed no mutagenic properties in the mammalian spermatogonial chromosome aberration test of the rat at the two tested sampling times of 24 hours and 48 hours. In the same system, Mitomycin C (positive reference item) induced significant damage.

Executive summary:

Dicyclohexylamine was assayed in an in vivo mammalian spermatogonial chromosome aberration test in the rat for the detection of damage to the chromosomes employing three dose levels.

The dose levels had been selected based on a range-finding study employing two male animals per dose. Three dose levels of 125, 250 and 500 mg Dicyclohexylamine/kg b.w. were tested. A dose level of 125 mg/kg b.w. revealed slight signs of toxicity 60 minutes after administration. 250 mg Dicyclohexylamine/kg b.w. caused slight to moderate signs of toxicity 15 minutes to 3 hours after administration. The high dose level of 500 mg/kg b.w. revealed slight to severe signs of toxicity 15 to 60 minutes and death within 3 hours after administration. The administration volume was 20 mL/kg b.w..

For the main study three ascending doses of 62.5, 125 or 250 mg Dicyclohexylamine/kg b.w., p.o. were administered. Further groups received the vehicle (sesame oil) and one further group the positive reference item Mitomycin C (5 mg/kg b.w., i.p.). Each group consisted of 7 male rats.

No signs of systemic toxicity were noted at 62.5 mg/kg b.w.. A dose level of 125 mg/kg b.w. revealed slightly reduced motility, slight ataxia and slightly reduced muscle tone 60 minutes after administration. The high dose level of 250 mg/kg b.w. revealed slightly to moderately reduced motility, slight to moderate ataxia, slightly to moderately reduced muscle tone and slight to moderate dyspnoea 15 minutes to 6 hours as well as slight to severe tonic convulsions 30 minutes to 6 hours and slight pilo-erection 30 minutes to 3 hours after administration (sacrifice after 24 or 48 hours).

In each group, the chromosome preparations from only five animals were used for evaluation. Two (2) sampling times were employed in this study: 24 hours after administration, samples were prepared from the negative reference item, positive reference item and all 3 doses of test item-treated animals; 48 hours after administration, samples were prepared only from vehicle and high dose-treated animals.

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Dicyclohexylamine ranged from 0.8% to 1.9%. These results were within the normal range, and no significant difference was observed compared to negative control (0.7% or 1.1%).

The percentage of cells with gaps was also within the range of the negative control (treated groups: 2.1% to 3.3%; control group: 1.8% to 3.9%).

No reduction of the mean mitotic index of the test item-treated animals per group compared with the mean mitotic index of the negative control was noted. The positive control, Mitomycin C, induced significant levels of chromosomal aberrations.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

2009-12-15 - 2010-04-10

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: micronucleus assay

Specific details on test material used for the study:

Name of test material (as cited in study report): Dicyclohexylamine- Physical state: colourless-yellowish liquid- Analytical purity: 99.45 %- Lot/batch No.: 89075804- Storage condition of test material: in supplied metal container at room temperature- stability/Expiration date: 6 months/04/2010

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic- Age at study initiation: 6-7 weeks- Weight at study initiation: 270-285 g- Assigned to test groups randomly: yes- Housing: cages with sterilized shavings of soft wood- Diet (e.g. ad libitum): ad libitum - Water (e.g. ad libitum): ad libitum- Acclimation period: at least 5 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22 + 3°C, permanently monitored- Humidity (%): 30 – 70 %, permanently monitored- Air changes (per hr): approximately 15 air changes per hour- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water for injections
- Amount of vehicle (if gavage or dermal): The volume of the application form at all dose levels was constant - 1 ml/100 g of body weight by adequate adjusting the concentration
- Lot/batch no. (if required): 0102190809

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The volume of the application form at all dose levels and negative control was constant - 1 ml/100 g of body weight by adequate adjusting the concentration. The animals were weighed before application. The application form of the test substance was prepared immediately before administration.

Duration of treatment / exposure:

24 and 48 hr

Frequency of treatment:

one single administration

Post exposure period:

24 and 48 hr

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

cyclophosphamide monohydrate 20 mg/kg
time interval: 24 hours
5 males and 5 females

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

Bone marrow cells were obtained from the femora immediately after euthanasia of animals. After excising and careful cleaning of the bone both femur ends were clipped off. Marrow
was gently flushed from the bone by 1 ml of bovine serum into the tube. Acquired bone marrow was several times mixed by syringe with thin needle.
The bone marrow with serum in tubes was centrifuged (5 min - 1000 rpm). The supernatant was gently removed, one drop of bovine serum was added to the sediment and this cell
suspension was mixed on mixer.
Clean and degreased slides were marked by the number of study, number of animal, sex and dose level. One drop of cell suspension was placed onto the slide and a smear was prepared
using a pusher slide. Two bone marrow smears were prepared per animal .
After drying (20 minutes, 60°C) the smears were fixed by ethanol - 5 minutes. Then they were twice rinsed by distilled water and stained by 5% solution of Giemsa - 15 minutes.
Before examination, the slides were coded.
Stained smears were examined by light microscope. 200 erythrocytes were evaluated per animal for the proportion of immature (polychromatic) and mature (normochromalic) erythrocytes („cytotoxicity index"-) in bone marrow.
At least 2000 polychromatic erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes.

Evaluation criteria:

Number of immature erythrocytes with micronuclei

Statistics:

The statistical analysis was performed by the ANOVA test - Analysis of Variance (software QC.Expert 2.5, Trilobyte, Statistical Software, Czech Rep.). Each of treatment groups was confronted with negative control group.

Key result

Sex:

male/female

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY:
In a pilot experiment a number of 6 animals - females were used in the two treatment groups:
1) 300 mg/kg of body weight -- exposition - 24 h: 3 animals
2) 200 mg/kg of body weight - exposition - 24 h: 3 animals
3. 7. 2. Clinical observation
All of 3 animals after administration of the dose 300 mg/kg lay on tlank and had very heavy symptoms of toxicity - heavy tremor of the body, piloerection, and anaemic skin. Two animals died one hour after application.
No symptoms of toxicity were observed in animals of the lowest dose level - 200 mg/kg.
MAIN STUDY:
The count of polychromatic erythrocytes with micronuclei after 24-hour and 48-hour exposition was raised with increasing dose level in both males and females. Statistically significant increase in the count of micronucleated immature erythrocytes compared to the control was recorded at the highest dose level of 200 mg/kg and middle dose level 100 mg/kg.

Conclusions:

The test substance, Dicyclohcxylamine was tested for the assessment of cytogenetic damage in vivo, using laboratory rat (Wistar). The study is a part of the test substance health hazard evaluation.
The test was performed according to the EU Method B.12, Mutagenicity -In vivo Mammalian Erythrocyte Micronucleus Test. The method is analogous to the OECD Test Guideline No. 474, Mammalian Erythrocyte Micronucleus Test.
The test substance was administered to animals by stomach tube in single dose. Three dose levels were chosen according to the results of pilot experiment - 200, 100 and 50 mg/kg of body weight. Two bone marrow sampling intervals were used - 24 and 48 hours after administration. The group of animals without administration and concurrent negative and positive controls were included. Each experimental group consisted of five males and five females. The smears obtained from bone marrow were examined by light microscope.
In the test group of males and females administered by the highest and middle dose levels (200 and 100 mg/kg/day). The test substance caused significantly increased frequency of immature erythrocytes with micronuclei in comparison with negative control animals.
Under the given test conditions, the test results were considered positive by the study authors.
A thorough re-evaluation of the individual study data does not provide convincing support for this evaluation, because:
o Strong cytotoxicity (without any dependence on the dose applied) was observed at the mid and high dose after 24 hour`s exposure of males and females and also at the high dose after 48 hours. In all those cases where no cytotoxicity could be observed no elevated levels of micronuclei could be detected.
Obviously has the marker system been damaged to the extent that no reliable signal for mutagenic activity can be gained from it.
o Cytotoxicity obviously reached a plateau already at the mid dose level at around 30 % reduction of cell integrity/viability which is not in line with the prerequisites of the respective OECD Guideline 474 where only the high dose should exhibit a depression of the ratio of polychromatic to normochromatic erythrocytes between at around 30 and 50 %.
In conclusion, the results of this assay are not thought to reliably indicate for potency of the substance Dicyclohexyamine for causing chromosomal aberrations.

Executive summary:

The test substance, Dicyclohcxylamine was tested for the assessment of cytogenetic damage in vivo, using laboratory rat (Wistar). The study is a part of the test substance health hazard evaluation.

The test was performed according to the EU Method B.12, Mutagenicity -In vivo Mammalian Erythrocyte Micronucleus Test. The method is analogous to the OECD Test Guideline No. 474, Mammalian Erythrocyte Micronucleus Test.

The test substance was administered to animals by stomach tube in single dose. Three dose levels were chosen according to the results of pilot experiment - 200, 100 and 50 mg/kg of body weight. Two bone marrow sampling intervals were used - 24 and 48 hours after administration. The group of animals without administration and concurrent negative and positive controls were included. Each experimental group consisted of five males and five females. The smears obtained from bone marrow were examined by light microscope.

In the test group of males and females administered by the highest and middle dose levels (200 and 100 mg/kg/day). The test substance caused significantly increased frequency of immature erythrocytes with micronuclei in comparison with negative control animals.

Under the given test conditions, the test results were considered positive by the study authors.

A thorough re-evaluation of the individual study data does not provide convincing support for this evaluation, because:

 o   Strong cytotoxicity (without any dependence on the dose applied) was observed at the mid and high dose after 24 hour`s exposure of males and females and also at the high dose after 48 hours. In all those cases where no cytotoxicity could be observed no elevated levels of micronuclei could be detected. 

Obviously has the marker system been damaged to the extent that no reliable signal for mutagenic activity can be gained from it. 

o   Cytotoxicity obviously reached a plateau already at the mid dose level at around 30 % reduction of cell integrity/viability which is not in line with the prerequisites of the respective OECD Guideline 474 where only the high dose should exhibit a depression of the ratio of polychromatic to normochromatic erythrocytes between at around 30 and 50 %. 

In conclusion, the results of this assay are not thought to reliably indicate for potency of the substance Dicyclohexyamine for causing chromosomal aberrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Conclusion on mutagenic potency of the substance Dicyclohexylamine

 

The following statement is based on:

-             (1) Report No. 10-104: Dicyclohexylamine, Mutagenicity – In vivo Mammalian Erythrocyte Micronucleus Test - Výzkumný ústav organických syntéz a.s., Centre of Ecology, Toxicology and Analytics, Rybitví 296, 533 54 Rybitví, Czech Republic

-             (2) Report No. 30530: IN VIVO MAMMALIAN SPERMATOGONIAL CHROMOSOME ABERRATION TEST OF DICYCLOHEXYLAMINE IN RATS BY ORAL ADMINISTRATION - according to OECD guideline 483 and EC No. 440/2008 Method B.23 -LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Redderweg 8, D-21147 Hamburg

With regard to mutagenic potency, literature data, not shown here, and test data according to (1) and (2) for the substance Dicyclohexylamine reveal the following:

1.           Consistently assays for point mutations in bacteria (so-called Ames Test) were negative with and without the addition of S9-mix (metabolic activation system), i.e. according to this in vitro system Dicyclohexylamine is not able to adversely affect respective genetic structures and to induce point mutations.

2.           Data from in vitro testing for chromosomal aberrations in Chinese hamster lung cells (CHL) give equivocal positive results at high concentrations and short exposure time as far as can be seen from the respective publication (i.e. at time point of 6 hours with S9-mix at 0.6 mg/mL and without S9-mix at 0.8 and 1.0 mg/mL).

From this test, no conclusive evaluation can therefore be drawn with regard to potency of the substance Dicylohexylamine for chromosomal aberrations.

3.           Testing results for in vivo detection of micronuclei in bone marrow (micronucleus assay) of rats according to (1) were considered positive by the study authors.

A thorough re-evaluation of the individual study data does not provide convincing support for this evaluation, because:

o            Strong cytotoxicity (without any dependence on the dose applied) was observed at the mid and high dose after 24 hour`s exposure of males and females and also at the high dose after 48 hours. In all those cases where no cytotoxicity could be observed no elevated levels of micronuclei could be detected.

Obviously has the marker system been damaged to the extent that no reliable signal for mutagenic activity can be gained from it.

o            Cytotoxicity obviously reached a plateau already at the mid dose level at around 30 % reduction of cell integrity/viability which is not in line with the prerequisites of the respective OECD Guideline 474 where only the high dose should exhibit a depression of the ratio of polychromatic to normochromatic erythrocytes between at around 30 and 50 %.

 

In conclusion, the results of this assay are not thought to reliably indicate for potency of the substance Dicyclohexyamine for causing chromosomal aberrations.

4.           Further testing of the substance Dicyclohexylamine for its potency to induce chromosomal aberrations facilitated the respective in vivo spermatogonial assay in rats after oral administration.

From the test results it can be concluded, that:

o            The substance did not induce any mutagenic events with regard to damage to chromosomes for up to the highest maximum tolerated dose (MTD).

o            The MTD did produce clear signs of systemic toxicity without to affect the mitotic index negatively, i.e. the substance Dicyclohexylamine has presumably reached the target cells at high enough, potentially adversely effective concentrations.

In conclusion, these in vivo test results do reliably and conclusively indicate for lack of potency of the substance Dicyclohexylamine to cause chromosomal aberrations.

The substance Dicyclohexylamine is devoid of toxicologically relevant mutagenic potency.

Since the substance octanoic acid, compound with dicyclohexylamine (1:1)

decomposes to dicyclohexylamine and octanoic acid and both dissociation products showed no mutagenic activity in various test systems it can be concluded that

octanoic acid, compound with dicyclohexylamine (1:1) is devoid of mutagenic properties.