Diisodecyl sebacate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

“In domain” QSAR models (both from the Danish EPA DB) applied to diisodecyl sebacate (DIDS) using the OECD QSAR Toolbox (OECD, 2012) predicted the following:

• DIDS was predicted to be negative in the Salmonella reverse mutation assay (Ames test), without in vitro DNA reactivity (Ashby fragments). No structural alerts were identified for DNA binding (by OASIS v.1.1 or OECD) or Ames mutagenicity (by OASIS v.1.1 or ISS).
• DIDS was predicted to show no in vitro DNA reactivity (Ashby fragments) and a negative result in the HGPRT forward mutation assay. No structural alerts were identified for DNA binding were identified by OASIS v.1.1 or OECD.
• DIDS was predicted to show no induction of unscheduled DNA synthesis in rat hepatocytes, and no in vitro DNA reactivity (Ashby fragments). An equivocal result was predicted for chromosome aberrations in Chinese hamster ovary cells, while a sister chromatid exchange assay [possibly in vivo] and a Syrian hamster embryo cell assay [possibly looking at transformation] were both predicted to be negative. No structural alerts were identified for DNA binding (by OASIS v.1.1 or OECD), micronucleus induction or chromosome aberration (by OASIS v.1.1).
• DIDS was predcted to show no DNA reactivity (Ashby fragments), and negative results in the rodent dominant lethal assay, the mouse micronucleus test, and the drosophila sex-linked recessive lethal assay. A negative result was also predicted in the sister chromatid exchange assay [apparently in vitro, according to the QSAR Toolbox, but possibly in vivo in mouse bone marrow, according to the Danish EPA DB site (Danish EPA, 2005)]. No structural alerts were identified for DNA binding were identified by OASIS v.1.1 or OECD, and no structural alerts for micronucleus induction or chromosome aberration were identified (by OASIS v.1.1), although an alert for in vivo micronucleus induction was detected by ISS (H-acceptor-path3-H-acceptor).

Link to relevant study records

Reference

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:

weight of evidence

Study period:

March 26 - April 18, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study on a read-across compound has been performed according to OECD and EC guidelines and to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test: the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate.
The selected treatment-levels for the main test were: 312.5, 625, 1250, 2500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item DIDA was dissolved in ethanol and ethanol is accepted and approved by
authorities and international guidelines.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9

Migrated to IUCLID6: (1 μg/plate for TA1535 and TA100)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9

Migrated to IUCLID6: (50 μg/plate for TA1537)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9

Migrated to IUCLID6: (0.5 μg/plate for TA98)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Migrated to IUCLID6: (0.5 μg/plate for TA102)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Anthramine (2 μg/plate for TA1535, TA1537, TA98 and TA100)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Anthramine (10 μg/plate for TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation (second experiment with S9-mix only)

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: one plate/dose-level in strain TA98, TA100 and TA102
- Mutagenicity experiments: three plates/dose-level in strain TA1535, TA1537, TA98, TA100 and TA102

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

Acceptance criteria:
This study is considered valid if the following criteria are fully met:
- The number of revertants in the vehicle controls is consistent with our historical data
- The number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data

Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the numberof revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a
positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Preliminary toxicity test: A moderate emulsion was noted in the Petri plates when scoring the revertants at dose-levels ≥ 1000 μg/plate.
Mutagenicity experiments: A moderate emulsion was noted in the Petri plates when scoring the revertants at dose-levels ≥ 1250 μg/plate.

RANGE-FINDING/SCREENING STUDIES:
No noteworthy toxicity was noted towards the three strains used, with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria, see section evaluation cirteria.
The study was therefore considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No noteworthy toxicity was noted towards all the strains used, both with and without S9 mix.
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Conclusions:

Interpretation of results (migrated information):
negative

In a guideline study, DIDA did not show mutagenic activity in the bacterial reverse mutation test with five Salmonella typhimurium strains when tested at up to 5000 μg/plate, with and without S9

Executive summary:

In a GLP study conducted in accordance with OECD Guideline 471, the mutagenicity of DIDA (a structurally-related read-across compound for DIDS) was assessed in a bacterial reverse mutation assay (“Ames” test).

 

Triplicate cultures of Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were incubated with DIDA at up to 5000 μg/plate for 48-72 hours (the pre-incubation period was 30 minutes) with and without metabolic activation by rat liver S9. Both the plate incorporation and pre-incubation methods were used (although the pre-incubation method was not conducted in the presence of S9).

 

There was no statistically significant increase seen in the number of revertant colonies in cultures treated with DIDA. Positive and vehicle controls performed as expected. DIDA exhibited no evidence of being mutagenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro gene mutation studies in bacteria

No standard bacterial gene mutation studies were available on DIDS.

 

“In domain” QSAR models (both from the Danish EPA DB) applied to DIDS using the OECD QSAR Toolbox (OECD, 2012) predicted DIDS to be negative in the Salmonella reverse mutation assay (Ames test), without in vitro DNA reactivity (Ashby fragments). No structural alerts were identified for DNA binding (by OASIS v.1.1 or OECD) or Ames mutagenicity (by OASIS v.1.1 or ISS).

Relevant read-across data:

A good quality GLP study, conducted in accordance with OECD Guideline 471, found no evidence of mutagenicity when DIDA was tested, with and without metabolic activation (S9), on triplicate cultures of Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 for 48-72 hr at up to 5000 μg/plate. Both the plate incorporation and pre-incubation (with a pre-incubation period of 30 minutes) methods were used (although the pre-incubation method was not performed without S9) (Haddouk, 2002a).

 

A similar study, equivalent to that described by OECD Guideline 471 (with GLP not mentioned), found no evidence of mutagenicity when DEHS was tested, with and without S9, on triplicate cultures of S. typhimurium strains TA98, TA100, TA1535 and TA1537. A pre-incubation period of 20 minutes was followed by an incubation period of 48 hr (Zeiger et al. 1985). DEHS was also not mutagenic in another apparently reliable Ames test on S. typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2uvrA/pKM101 at concentrations of up to 5000 μg/plate, with and without S9 (JETOC, 2008).

 

In vitro gene mutation study in mammalian cells

No standard gene mutation assays were available on DIDS in mammalian cells.

 

“In domain” QSAR models (all from the Danish EPA DB) applied to DIDS using the OECD QSAR Toolbox (OECD, 2012) predicted no in vitro DNA reactivity (Ashby fragments) and a negative result in the HGPRT forward mutation assay. No structural alerts were identified for DNA binding were identified by OASIS v.1.1 or OECD.

 

Relevant read-across data:

A good quality GLP study, conducted in accordance with OECD Guideline 476, found equivocal evidence of mutagenicity when DIDA was tested on duplicate cultures of mouse lymphoma (L5178Y) cells for 3 hr (with S9 and without S9) or for 24 hr (without S9 only). No statistically significant increase in mutation frequency was seen following 3 hr of treatment, but results for the 24-hr treatments were equivocal, with 2-fold increases in mutation frequency seen at concentrations also exhibiting high levels of cytotoxicity. Slight increases were seen in the mutation frequency of cultures treated with 750 or 875 μg/ml (1.5 and 1.6-fold, respectively), and were again seen with significant cytotoxicity – relative survival was 13 and 8% of that seen for vehicle control. An increased number of small colonies were also observed at concentrations of at least 500 μg/ml (possibly indicating the occurrence of gross chromosome aberrations). Overall, results from this experiment were considered equivocal (Haddouk, 2002b).

 

In vitro cytogenicity study in mammalian cells or in vitro micronucleus study

No standard cytogenicity or micronucleus assays were available on DIDS in mammalian cells.

 

“In domain” QSAR models (all from the Danish EPA DB) applied to DIDS using the OECD QSAR Toolbox (OECD, 2012) predicted no induction of unscheduled DNA synthesis in rat hepatocytes, and no in vitro DNA reactivity (Ashby fragments). An equivocal result was predicted for chromosome aberrations in Chinese hamster ovary cells, while a sister chromatid exchange assay [possibly in vivo] and a Syrian hamster embryo cell assay [possibly looking at transformation] were both predicted to be negative. No structural alerts were identified for DNA binding (by OASIS v.1.1 or OECD), micronucleus induction or chromosome aberration (by OASIS v.1.1).

 

Relevant read-across data:

In a good quality GLP study, conducted in accordance with OECD Guideline 473, DIDA was incubated with duplicate cultures of human lymphocytes for 3 hr (with and without S9), or 20 or 44 hr (without S9 only) at up to 2495 μg/ml. Following cell cycle arrest, 200 metaphase spreads per dose were examined for chromosome aberrations. No chromosome aberrations were seen following the 3-hr treatment, either with or without S9. A reproducible and statistically significant increase in the frequency of cells with chromosome aberrations was seen at the top concentration only following the 20 or 44-hr treatments. Cytotoxicity was, however, also observed following these longer treatment times (described as "suspected to be highly toxic" at the top concentration) (Haddouk, 2002c).

 

It has also been noted that the procedure given in Haddouk (2002c) deviates from the current protocol by collecting blood from two supposedly healthy subjects, instead of one. The lymphocytes from the male subject reacted, but those of the female did not. Possible reasons include an unhealthy male donor, currently undergoing therapy, viral exposure within the 6 months prior to having his blood drawn, etc. As the male was reported to be healthy, helpful results could be obtained if the experiment was repeated.

Genotoxicity (in vivo)

No standard in vivo assays were available for DIDS (or the read-across compounds DIDA, DEHS or DOS).

 

“In domain” QSAR models (all from the Danish EPA DB) applied to DIDS using the OECD QSAR Toolbox (OECD, 2012) predicted no DNA reactivity (Ashby fragments), and negative results in the rodent dominant lethal assay, the mouse micronucleus test, and the drosophila sex-linked recessive lethal assay. A negative result was also predicted in the sister chromatid exchange assay [apparently in vitro, according to the QSAR Toolbox, but possibly in vivo in mouse bone marrow, according to the Danish EPA DB site (Danish EPA, 2005)]. No structural alerts were identified for DNA binding were identified by OASIS v.1.1 or OECD, and no structural alerts for micronucleus induction or chromosome aberration were identified (by OASIS v.1.1), although an alert for in vivo micronucleus induction was detected by ISS (H-acceptor-path3-H-acceptor).

QSAR Analyses - DIDS

As no studies are available for DIDS, a read-across approach has been applied from analagous substances. This read-across approach has been previously described in the dossier. To strengthen this approach, DIDS and other substances were profiled in the OECD QSAR toolbox, using predictions to determine the outcomes for genetic toxicity endpoints. The results of these are described in the table below:

Substance identity
CAS number	28473-19-0	2432-87-3	122-62-3	27178-16-1
Chemical name	DIDS	DOD	DEHS	DIDA
SMILES	CC(C)CCCCCCCOC(=O)CCCCCCCCC(=O)OCCCCCCCC(C)C	CCCCCCCCOC(=O)CCCCCCCCC(=O)OCCCCCCCC	CCCCC(CC)COC(=O)CCCCCCCCC(=O)OCC(CC)CCCC	CC(C)CCCCCCCOC(=O)CCCCC(=O)OCCCCCCCC(C)C
Profilers
General Mechanistic
DNA binding by OASIS	No alert found	No alert found	No alert found	No alert found
DNA binding by OECD	No alert found	No alert found	No alert found	No alert found
DNA alerts for CA and MNT by OASIS	No alert found	No alert found	No alert found	No alert found
in vivo mutagenicity (Micronucleus) alerts by ISS	No alert found	No alert found	No alert found	No alert found
DNA alerts for AMES by OASIS	No alert found	No alert found	No alert found	No alert found
in vitro mutagenicity (Ames test) alerts by ISS	No alert found	No alert found	No alert found	No alert found

The QSAR analyses suggest that the substances are not genotoxic, which is corroborated by the existing studies on the analogue substances.

References

ACToR (2012). Aggregated Computation Toxicology Resource of the United States Environmental Protection Agency. Accessed June 2012.

Danish EPA (2005). Danish Environmental Protection Agency. User Manual for the Internet Version of the Danish (Q)SAR Database. Database Version 1 May 2005.http://130.226.165.14/User_Manual_Danish_Database.pdf

JETOC (2008). Japan Chemical Industry Ecology-Toxicology & Information Center. Mutagenicity test data of existing chemical substances based on the toxicity investigation system of the industrial safety and health law, Supplement 4 (cited in ACToR).

Justification for selection of genetic toxicity endpoint
OECD guideline study conducted to GLP on a read-across compound.

Justification for classification or non-classification

The weight of evidence suggests that no mutagenic potential or DNA reactivity was predicted for DIDS in the QSAR analysis. Reliable in vitro assays with bacteria (Ames tests) found no evidence of mutagenic activity for DOS or DIDA. A slight increase in mutation frequency was seen in mouse lymphoma cells exposed to DIDA for 24 hr (but not after 3 hr), but these exposures were associated with significant cytotoxicity. An increase in chromosome aberrations was seen in cultured human lymphocytes exposed to DIDA for 20 or 44 hr (but not after 3 hr), but these longer treatment times were again associated with cytotoxicity. Neither DOS nor DIDA is classified for mutagenicity.

On this basis, the weight-of-evidence indicates that DIDS does not require classification as a mutagen according to the criteria of the EU CLP Regulation (EC) 1272/2008 or the Dangerous Substances Directive 67/548/EEC.