Trimethyl orthoacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Trimethyl orthoacetate (TMOA)

- in vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): 50 - 5000 µg/plate, Salmonella typhimuriumTA 1535, TA 1537, TA 98 and TA 100: negative with and without metabolic activation (OECD TG 471)

Trimethyl orthoacetate is rapidly hydrolysed to methanol (CAS no. 67 -56 -1) and methyl acetate, which further hydrolyses to acetic acid (CAS no. 64 -19 -7) as final reaction product in the presence of water or moisture at pH 4, 7 and 9 (< 2.4 h at 50°C). Under neutral (pH 7) and acidic (pH 4) conditions, the half-life was < 1 h. Accordingly, reliable data of the hydrolysis products methanol and acetic acid are used for the assessment of the genotoxic potential beside one gene mutation study in bacteria on Trimethyl orthoacetate, which is entirely appropriate to draw conclusions on the genetic toxicity of Trimethyl orthoacetate.

1. Methanol:

- In vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100: negative with and without metabolic activation, S. typhimurium TA 1538: negative with and without metabolic activation, E. coli WP2 uvr A: negative with and without metabolic activation (OECD TG 471)

- In vitro: Gene mutation (mammalian cell gene mutation assay (HPRT)): Chinese hamster lung fibroblast (V79): negative with and without metabolic activation (equivalent to OECD TG 476)

- In vitro: Chromosome abberation (mammalian cell micronucleus test): Chinese hamster lung fibroblast (V79): negative with and without metabolic activation (test procedure in accordance with accepted standard methods)

2. Acetic acid

- In vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): Salmonella typhimurium TA 100, TA 1535, TA 97 and TA98: negative with and without metabolic activation (equivalent to OECD TG 471)

- In vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): Salmonella typhimurium TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98: negative with metabolic activation (equivalent to OECD TG 471)

- In vitro: Gene mutation (mammalian cell gene mutation assay): mouse lymphoma L5178Y cells: inconclusive with and without metabolic activation, not indicating any significant genotoxic or mutagenic activity in mammalian cells (equivalent to OECD TG 476)

- In vitro: Chromosome abberation (mammalian chromosome abberation test): Chinese hamster ovary cells (CHO): negative with and without metabolic activation (equivalent to OECD TG 473)

- In vitro: Chromosome abberation (mammalian chromosome abberation test): Chinese hamster lung fobroblast (V79): negative with and without metabolic activation (OECD TG 473)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-07-1995 - 21-07-1995 (experimental phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well-documented study according to OECD 471 with minor deviations: only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD-Guideline for the Testing of Chemicals "Genetic Toxicology: Salmonella typhimurium, Reverse Mutation Assay", Method No. 471 (OECD, adopted May 1983)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

"Teil B der 17. Anpassung der Richtlinie 67/548/EWG an den technischen Fortschritt. B.14 Mutagenität"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his (S. typhimurium)

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. Bruce Ames, University of California, Berkeley, U.S.A..
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD, EC).

Additional strain / cell type characteristics:

other: see "Any other information on materials and methods"

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male Wistar rat S9

Test concentrations with justification for top dose:

Five dose levels of Trimethylorthoacetate ranging from 50 to 5000 pg/plate in the absence and in the presence of metabolic activation were spaced at half-log intervals.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

Solvent controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (for test item, positive control, 100 µl/plate (plate incorporation), 50 µl/plate (preincubation))

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, 1st experiment); preincubation (2nd experiment)
- Cell density at seeding (if applicable): 0.1 ml of a fresh bacterial culture (10exp9 cells/ml)

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EC).

Evaluation criteria:

For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.

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

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

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

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

Positive controls validity:

valid

Additional information on results:

A reduced growth of the bacterial background lawn, indicative of test compound induced cytotoxicity, was not detectable with any of the tester strains.
All four bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolic activation by S9 mix. Negative (solvent) controls were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable.
In both experiments, no indication of test compound induced mutagenicity was observed with either one of the four tester strains TA 98, TA 100, TA 1535, and TA 1537, with or without metabolic activation.
All criteria for a valid study were met as described.

Results and Discussion

Titers of the overnight cultures

As shown in the following table, all bacterial titers of the overnight cultures were in excess of 10⁸ bacteria/ml.

Titers of overnight cultures (bacteria/mililiter)

	TA 98	TA 100	TA 1535	TA 1537
Plate incorporation test	1.7x10^9	1.7x10^9	1.0x10^9	8.6xl0^8
Preincubation test	1.6x10^9	1.1x10^9	1.4x10^9	1.3xl0^9

Conclusions:

Interpretation of results: negative with and without metabolic activation

The study was performed according to the OECD Guideline 471 with deviations (no E.coli WP2 strains was tested) according to the principles of the good laboratory practice and therefore considered to be of high quality (reliability Klimisch 2). The vehicle and the positive control substances fulfilled validity criteria of the test system. In conclusion , the results indicate that Trimethyl orthoacetate did not induce a mutagenic effect at concentrations ranging from 50 to 5000 µg/plate in the four S. typhimurium tester strains TA 1535, TA 1537, TA 98 and TA 100.

Executive summary:

The test substance Trimethyl orthoacetate (TMOA) was investigated under GLP according to OECD TG 471 in the Salmonella/microsome test for point mutations using four Salmonella typhimurium LT2 mutants. These tester strains were the histidine auxotrophic strains TA 1535, TA 1537, TA 98 and TA 100. Concentrations ranging from 50 to 5000 µg per plate were employed. The solvent was DMSO.

No bacteriotoxic effects were caused at the concentrations ranging from 50 to 5000 µg/plate. The total number of viable cells, the number of revertants and the background growth were within the normal range. No evidence of mutagenic activity of TMOA was found. No biologically relevant or dose-related increase in reversion rate was observed, neither with nor without S 9 mix in comparison to the negative control (solvent). The sensitivity of the test system was demonstrated by the marked mutagenic effects exerted by the positive controls 9-aminoacridine, sodium azide, 2-nitrofluorene and 2-aminoanthracene.

In summary, it may be concluded that the test substance TMOA caused no mutagenic effect at concentrations ranging from 50 to 5000 µg/plate.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon, Trp-operon (E. coli)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction of KC500-pretreated rats

Test concentrations with justification for top dose:

5, 10, 50, 100, 500, 1000, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

0.01 and 5.0 µg/plate, respectively

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) without S9, benzo(a)pyrene (B(a)P) with S9

Remarks:

TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

both 5.0 µg/plate, respectively

Positive control substance:

other: N-ethyl -N'-nitro-N-nitrosoguanidine (ENNG) without S9, 2-aminoanthracene (2AA) with S9

Remarks:

TA1535both 5.0 µg/plate, respectively

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

0.05 and 5.0 µg/plate, respectively

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) without S9, 2-aminoanthracene (2AA) with S9

Remarks:

WP2 uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

0.05 and 5.0 µg/plate, respectively

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) without S9, benzo(a)pyrene (B(a)P) with S9

Remarks:

TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

80.0 and 5.0 µg/plate, respectively

Positive control substance:

other: 9-aminoacridine (9AC) without S9, benzo(a)pyrene (B(a)P) with S9

Remarks:

TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

0.25 and 5.0 µg/plate, respectively

Positive control substance:

other: 4-nitroquinoline-1-oxide (4NQO) without S9, benzo(a)pyrene (B(a)P) with S9

Remarks:

TA1538

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition of revertant clones

Evaluation criteria:

Doubling of revertant numbers in comparison to control and dose-response correlation.

Key result

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:

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:

E. coli WP2 uvr A

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

Additional information on results:

For the phrase selection in field 'Endpoint' the following source information was used as basis:
Type of genotoxicity: gene mutation
Type of study: bacterial reverse mutation assay (e.g. Ames test)
Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Species/strain (Method):
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- S. typhimurium TA 1538
- E. coli WP2 uvr A

Maximum number of revertants:

	Control (water) [mean±SD]		Test substance (µg/plate)
Strain	-S9	+S9	-S9	+S9
TA100	149±17.1	161±16.2	175 (100)	180 (50)
TA1535	28±6.9	15±3.6	35 (50)	17 (5000)
TA98	29±6.2	39±8.6	42 (50)	39 (1000)
TA1537	16±6.4	21±8.1	22 (5000)	35 (5)
TA1538	21±5.5	28±7.0	18 (500)	31 (5)
E.coli WP2 uvrA	32±7.3	33±10.3	36 (5000)	43 (10)

The test substance was not mutagenic in the tested strains up to 5000 µg/plate.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, also largely meeting current standards, sufficient documentation, acceptable for assessment.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Exposure and expression period combined

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

no data

Test concentrations with justification for top dose:

15.8, 31.7, 47.4, 63.3 mg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (Eagle's MEM)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with S9 Migrated to IUCLID6: 1 and 2 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: MNNG, 0.29 and 0.59 µg/mL

Remarks:

without S9

Details on test system and experimental conditions:

DURATION
- Exposure duration: 6 days
- Expression time (cells in growth medium): combined with 6 days exposure
- Selection time (if incubation with a selection agent): after 6 days

SELECTION AGENT (mutation assays): 8-Azaguanin, 6-Thioguanin, Ouabain

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (colony formation)

Evaluation criteria:

Significant increase in V79 cells resistant to 8-Azaguanine, 6-Thioguanine or Ouabain.

Statistics:

Calculation of mean mutation frequency±S.D. per 10e6 surviving cells.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

63.3 mg/ml (approx. 70 % inhibition of colony formation)

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

The test substance induced no increases in mutant frequency in gene mutation to drug resistance vs. negative control, whereas the positive control DMN produced increases in dose-related manner in the presence of metabolic activation (S9 mix), and MNNG in the absence of metabolic activation.

Maximum mutation frequency of V79 cells (per 10e6 survival cells, mean±SD) for resistance towards 6-TG, 8-AG and Ouabain after methanol treatment:

	Control		Test substance (mg/mL)
Selectant	-S9	+S9	-S9	+S9
6-Thioguanine	0.70±1.79	0.94±2.18	1.55±3.08 (47.4 mg/mL)	0.84±2.21 (31.7 mg/mL)
8-Azaguanine	23.42±8.70	24.29±7.30	22.34±7.39 (31.7 mg/mL)	20.05±13.01 (31.7 mg/mL)
Ouabain	1.23±2.23	0	2.64±2.79 (47.7 mg/mL)	0.11±0.36 (47.7 mg/mL)

Conclusions:

Interpretation of results (migrated information):
negative

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with accepted standard methods, sufficiently documented, acceptable for assessment.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

In vitro micronucleus test with V79 cells comparing alcohols, acetone and various alkylating agents.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

50 µL/mL (approx. 40 mg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: medium (Eagle's MEM + 10 % FCS), acetone for positive controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

0, 0.02, 0.04, 0.08 µg/mL; solvent acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

0, 0.4, 2, 10, 50, 100 µg/mL; solvent acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

0, 25, 50, 100 µg/mL; solvent acetone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 48 h

STAIN (for cytogenetic assays): 4% Giemsa

NUMBER OF REPLICATIONS: not reported

NUMBER OF CELLS EVALUATED: 7000 interphase cells at each concentration used

OTHER
Cells were plated at a density of 13000 cells/cm², incubated for 15-18 h, then treated with the test substance. After treatment, the cells were incubated for 48 h, then collected, subjected to hypotonic treatment with KCl, fixed with acetic acid-methanol, and then stained with 4% Giemsa.

Evaluation criteria:

Criteria used to score MN: (1) staining intensity equal to that of the nucleus, (2) diameter less than one-fifth that of the nucleus, (3) location in cytoplasm, (4) no contact with nucleus to distinguish from nuclear blebs.

Statistics:

The dose response was estimated by linearr regression curves. Difference in the sensitivity of mutagenic compounds was established by comparing the slopes of corresponding dose-response regression curves. For a comparison of mean values, Student's t test was used.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

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

No MN increases induced by any alcohol or acetone, whereas the alkylating agents produced significant MN frequencies above medium controls.

	Max. number of MN/1000 cells [mean±S.E.]
	Control (solvent dose)	Chemical (dose)
Methanol	4.00±0.71 (50 µL/mL)	3.50±1.19 (50 µL/mL)
MNNG	3.2±0.40 (5 µL/mL)	8.2±0.83 (0.08 µg/mL)
MMS	3.9±0.59 (5 µL/mL)	16.5±0.50 (100 µg/mL)
EMS	2.2±0.40 (5 µL/mL)	7.0±0.69 (100 µg/mL)

Solvent for methanol: medium

Solvent for MNNG, MMS, EMS: acetone

Conclusions:

Interpretation of results (migrated information):
negative

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non GLP, near guideline study, animal experimental study, published in peer reviewed literature, minor restrictions in design and/or reporting but otherwise adequate for assessment.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

not all the recommended bacterial strains used. Deviations from guideline do not affect the results presented.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

other: S. typhimurium TA100, TA1535, TA97 and TA98

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 from Aroclor-induced male Sprague-Dawley rats or male Syrian hamsters

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333, 6666 and 1000 µg/plate

Vehicle / solvent:

- distilled water

Negative solvent / vehicle controls:

yes

Remarks:

H2O

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 Migrated to IUCLID6: sodium azide TA1535 and TA100

Positive control substance:

9-aminoacridine

Remarks:

without S9 Migrated to IUCLID6: 9-aminoacridine TA97

Positive control substance:

other: 4-nitro-o-phenylenediamine TA98

Remarks:

without S9

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains, with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Expression time (cells in growth medium): 2 days

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

Evaluation criteria:

A chemical was judged mutagenic or weakly mutagenic if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a positive response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic if they did not meet the criteria for a mutagenic or questionable response.

Species / strain:

other: S. typhimurium TA100, TA1535, TA97 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Slight clearing of background lawn was seen with 30% hamster and rat S9 at doses of 6666.0 and 10000.0 µg/plate in all the bacterial strains.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Acetic acid was not mutagenic to S. typhimurium strains TA100, TA1535, TA97 and TA98.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Minor restrictions in design and reporting but otherwise adequate for assessment

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

S.typh TA102, E.coli WP2 uvrA or E. coli WP2 uvrA (pKM101) were not used

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella Typhimurium TA92, TA1535, TA100, TA1537, TA94 and TA98

Metabolic activation:

with

Metabolic activation system:

S9 from polychlorinated biphenyls induced rat liver

Test concentrations with justification for top dose:

6 different concentrations up to a maximum dose of 10 mg/plate (the highest non-cytotoxic dose used in the experiment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: phosphate buffer

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The number of revertant (his+) colonies was scored after incubation at 37°C for 2 days. The result was considered positive if the number of colonies was twice the number in the control (exposed to the appropriate solvent). If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays were performed.

Species / strain:

other: Salmonella Typhimurium TA92, TA1535, TA100, TA1537, TA94 and TA98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested at a maximum non-cytogenic dose of 10 mg/plate

Vehicle controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation

Acetic acid was non-mutagenic in the Ames assay at concentrations of up to 10 mg/plate with metabolic activation.

Executive summary:

Acetic acid was non-mutagenic in the Ames assay at concentrations of up to 10 mg/plate with metabolic activation.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Near guideline studies, published in peer-reviewed literature, adequate for assessment

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells grown in Fischer's medium supplemented with 10% horse serum and 0.02% pluronic F-68
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes, at approximately 3 month intervals

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced liver S9 from male Sprague-Dawley rats

Test concentrations with justification for top dose:

0.04-0.3 µg/mL

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: 4.7 x 10-6 M (or methyl methanesulfonate at 10-20 µg/mL)

Positive control substance:

3-methylcholanthrene

Remarks:

Migrated to IUCLID6: 1.86 x 10-5 M (or dimethylbenz[α]-anthracene at 0.5-4 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

- A total of 1.2 x 107 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37± 1°C, washed twice with growth medium, and maintained at 37± 1°C for 48 h in log phase growth to allow recovery and mutant expression.
- Cells in the cultures were adjusted to 3 x 105/mL at 24 h intervals.
- They were then cloned (1 x 106 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar.
- Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. The 100x stock solution of TFT in saline was stored at -70°C and was thawed immediately before use.
- Plates were incubated at 37±1°C in 5% CO2 in air for 10-12 days and then counted with an automated colony counter. Only colonies larger than -0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106 surviving cells.

Evaluation criteria:

Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

other: Inconclusive. With and without S9

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see remarks on results

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Interpretation of results (migrated information):
other: Inconclusive. With and without metabolic activation

Acetic anhydride is considered to have no significant mutagenic activity in mammalian cells.

Executive summary:

Acetic anhydride has been examined for mutagenic activity in mammalian cells in vitro using the mouse lymphoma L5178Y assay. Although the data can be formally regarded as equivocal, they do not indicate any significant genotoxic activity for acetic anhydride in mammalian cells in vitro. This evaluation of the data reports the findings as inconclusive.

Acetic anhydride is considered to have no significant mutagenic activity in mammalian cells.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non GLP, near guideline study, animal experimental study, published in peer reviewed literature, minor restrictions in design and/or reporting but otherwise adequate for assessment

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

The study was targeted to examine the effect of pH changes induced versus the organic acids themselves

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Hams's F12 medium supplemented with kanamycin, 17mM NaHCO3 and 10% foetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

up to 20mM acetic acid

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

An aliquot of 50-75 µl of diluted acid was added to the culture medium (5 mL). In the absence of S9 mix, chromosome preparations were made by an air-drying method 24 hr after the addition of each organic acid. Cytotoxicity of each acid was also examined by counting surviving cells. In the presence of S9 mix, the cells were washed with physiological saline after a 6 hr treatment, and then incubated with fresh medium for 18 hr before the chromosome preparations were made (Ishidate, 1987).
In order to study the effect of neutralization of the treatment medium, 2 kinds of treatment media were examined; one was adjusted to pH 5.8 or pH 6.0 with each of these acids and the other was so adjusted then immediately neutralized to pH 6.4 and pH 7.2 with 1 M NaOH. In order to study the effect of enhancement of the buffering ability, chromosomal aberration tests were carried out on these acids in the absence of S9 mix using F12 medium Containing 34 mM NaHCO3 (twice the concentration usually employed). Furthermore, to study the effect of alteration of the buffering system, the tests were performed on these acids in the absence of S9 mix using F12 medium containing 30 mM HEPES instead of NaHCO3 as a buffer. This medium was adjusted to pH 8.5 with NaOH, and the cells were incubated in closed culture vessels.
The pH of the medium was measured initially and at 6 hr and 24 hr after the treatment using a pH meter. 100 well-spread metaphases from 1 experiment were observed at each point to record the percentage (including gaps) of cells with chromosomal aberrations. At least 2 independent experiments were carried out in each case.

Evaluation criteria:

100 well-spread metaphases from 1 experiment were observed at each point to record the percentage (including gaps) of cells with chromosomal aberrations. At least 2 independent experiments were carried out in each case.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at physiological pH

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 10mM

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: A dose-dependent increase in chromosomal aberrations was observed at concentrations of 10mM acetic acid in the absence of S9-mix and at 8mM acetic acid in the presence of S9 mix. Thee concentrations were close to the cytotoxic limit at which the cells could no longer be evaluated. These effects were abolished by neutralising the test medium or increasing the buffering capacity, and can therefore be attributed to the acidic pH of the incubation medium rather than a consequence of an intrinsic clastogenic potential of acetic acid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

TABLE 1 -CHROMOSOME ABERRATIONS IN CHO-KI CELLS INDUCED BY ACETIC ACID

INTHE ABSENCE OF S9 MIX
Dose (mM)	pH after treatment			Cells scored	Type and number of aberrations							Aberrant cells (%)^a
	initial	6 h	24 h		ctg	csg	ctb	csb	cte	cse	f
8	6.3	7.1	7.4	200	1	0	0	1	0	0	0	1.0
10	6.1	6.9	7.3	200	0	0	0	0	0	0	0	0
12	5.9	6.7	7.1	200	2	1	9	0	0	0	0	5.0
14	5.7	6.3	6.6	130	4	0	13	1	0	0	0	10.8
16	5.5	5.9	5.8	Toxic								Toxic

 

IN THE PRSENCE of S9
Dose (mM)	pH after treatment			Cells scored	Type and number of aberrations							Aberrantcells (%)^a
	initial	6 h	24 h		ctg	csg	ctb	csb	cte	cse	f
4	6.7	7.1	-	200	0	0	1	0	2	0	0	1.5
8	6.2	6.6	-	200	1	0	2	0	1	0	0	2.0
10	5.9	6.3	-	200	10	2	19	4	28	0	1	20.0
12	5.7	5.8	-	Toxic								Toxic

a All structural aberrations including gaps.

ctg, chromatid gaps; csg, chromosome gaps; ctb, chromatid breaks; csb, chromosome breaks; cte, chromatid exchanges; f, fragmentations; cse, chromosome exchanges including dicentric and ring chromosomes.

TABLE 2 -CHROMOSOME ABERRATIONS IN CHO-KI CELLS INDUCED BY ACETIC ACID UNDER CONDITIONS OF INCREASED BUFFEREING CAPACITY

F12 MEDIUM CONTAINING 34 mM NaHCO3INTHE ABSENCE OF S9 MIX
Dose (mM)	pH after treatment			Cells scored	Type and number of aberrations							Aberrant cells (%)^a
	initial	6 h	24 h		ctg	csg	ctb	csb	cte	cse	f
20	6.1	7.1	7.4	200	1	0	0	0	0	1	0	0.5
25	5.9	6.9	7.2	200	2	0	0	0	0	0	0	1.0
30	5.6	6.5	6.8	200	3	0	6	0	9	0	0	9.0

 

F12 MEDIUM CONTAINING 30 mM HEPES AS A BUFFER IN THE PRSENCE of S9
Dose (mM)	pH after treatment			Cells scored	Type and number of aberrations							Aberrantcells (%)^a
	initial	6 h	24 h		ctg	csg	ctb	csb	cte	cse	f
10	7.6	7.4	7.0	200	0	0	0	0	0	0	0	0
20	7.0	7.0	6.7	200	1	0	0	0	0	0	0	0.5
25	6.6	6.5	6.6	200	3	1	7	0	3	0	0	7.0
30	6.0	6.1	6.1	Toxic								Toxic

a All structural aberrations including gaps.

ctg, chromatid gaps; csg, chromosome gaps; ctb, chromatid breaks; csb, chromosome breaks; cte, chromatid exchanges; f, fragmentations; cse, chromosome exchanges including dicentric and ring chromosomes.

Conclusions:

Acetic acid was not clastrogenic to Chinese hamster ovary cells under test conditions that maintained the pH of the incubation medium in the physiological range.

Executive summary:

Using Chinese hamster ovary Kl cells, chromosomal aberration tests were carried out with acetic, formic and lactic acids and the relationship between the pH of the medium and the clastogenic activity was examined. All these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12-16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was observed.

Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8-6.0). However, it was found that about 10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM acetic acid (initial pH 7.0-7.1). In the initial pH range ofi.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Minor restrictions in design and reporting but otherwise adequate for assessment

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung fibroblast cell line CHL

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum Essential Medium (MEM;GIBCO) supplemented with 10% calf serum

Metabolic activation:

without

Test concentrations with justification for top dose:

3 different concentrations up to a maximum dose of 1 mg/mL (the highest non-cytotoxic dose used in the experiment). Maximum dose selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: physiological saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 and 48 hours
- Colcemid (final concentration 0.2 µg/mL) was added to the culture 2 hours before cell harvesting

STAIN: Giesma solution (1.5% at pH 6.8)

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases were observed microscopically

EXAMINATIONS: Incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate

Evaluation criteria:

Results considered to be negative if the incidence of aberrations was less than 4.9%, equivocal if it was between 5.0 and 9.9% and positive if it was more than 10%. If no reasonable dose-response relationship was found, additional experiments were carried out at similar dose levels.

Species / strain:

other: Chinese hamster lung fibroblasts CHL

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested at a maximum non-cytogenic dose of 1mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Maximum concentration tested 1.0 mg/mL

Polyploid 0%

Structural aberrations after 48 hours 3.0%

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

Acetic acid was negative in the chromosome aberration assay using Chinese hamster lung fibroblasts at concentrations of up to 1mg/mL without metabolic activation.

Executive summary:

Acetic acid was negative in the chromosome aberration assay using Chinese hamster lung fibroblasts at concentrations of up to 1mg/mL without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Trimethyl orthoacetate is rapidly hydrolysed to methanol (CAS no. 67 -56 -1) and methyl acetate, which further hydrolyses to acetic acid (CAS no. 64 -19 -7) as final reaction product in the presence of water or moisture at pH 4, 7 and 9 (< 2.4 h at 50°C). Under neutral (pH 7) and acidic (pH 4) conditions, the half-life was < 1 h. Accordingly, reliable data of the hydrolysis products methanol and acetic acid are used for the assessment of the genotoxic potential, which is entirely appropriate to draw conclusions on the genetic toxicity of Trimethyl orthoacetate.,

1. Methanol

- In vivo: Chromosome abberation (micronucleus assay): negative (equivalent to OECD 474; 4 concentrations (300, 600, 1200, 2500 mg/kg bw) intraperitoneal administered to male mice (Swiss Webster))

- In vivo: Chromosome abberation (micronucleus assay): negative (test procedure in accordance with accepted standard methods; 1 concentration (2500 mg/kg bw) orally administered to female mice ( CD-1))

2. Acetic acid

- In vivo: Chromosome abberation (micronucleus assay): negative (equivalent to EU Method B.12; 3 concentrations (1, 5, 20 mg/kg bw) administered to male and female rats (CD (Sprague-Dawley)) by inhalation (vapour))

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

- limited documentation

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss Webster

Sex:

male

Details on test animals or test system and environmental conditions:

no data

Route of administration:

intraperitoneal

Vehicle:

no data

Details on exposure:

no data

Duration of treatment / exposure:

4 days

Frequency of treatment:

no data

Post exposure period:

24 h

Remarks:

Doses / Concentrations:
300, 600, 1200, 2500 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes

Positive control(s):

Caffeine (-folate)
Urethane (+folate)

Tissues and cell types examined:

erythrocytes from blood samples

Details of tissue and slide preparation:

see any other information on materials and methods

Evaluation criteria:

no data

Statistics:

no data

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

see remarks on results

Folate limitation resulted in an about 15-20 times lower folate blood level than in the high-folate groups.

4/10 folate-deficient animals receiving 2500 mg/kg died between days 2 and 3.

No difference in micronucleus frequency (MN in PCEs) and in RNA-positive blood erythrocytes was seen between treated groups and controls while animals treated with the positive control substances responded as expected:

+folate: MN 0.17 - 0.23 % vs. 0.23 % in saline control

-folate: MN 0.31 - 0.37 % Vs. 0.38 % in saline control.

Caffeine (+folate): MN 0.34 %

Caffeine (-folate): MN 2.42 %

Urethane (+folate): MN 2.52 %.

The results indicate that methanol is nonclastogenic to the developing erythroblast in bone marrow and does not inhibit red blood cell production in either normal or folate-deficient mice.

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

The study was performed during a developmental investigation under folate-deficient and -sufficient conditions.
After oral methanol application, the frequency of MN was examined in maternal peripheral blood and fetal blood.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Inc.
- Weight at study initiation: 12-14 g
- Housing: Weanling mice were housed in stainless steel, wire-bottomed cages.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23 °C
- Humidity (%): 50 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

deionised water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
2.5 g/kg (15.65 % Optima HPLC grade MeOH in deionised water)

Duration of treatment / exposure:

gestation day 6 through 10 during pregnancy

Frequency of treatment:

twice daily

Post exposure period:

48 h after the final exposure: maternal peripheral blood was collected
gestation day 18: fetal blood was taken

Remarks:

Doses / Concentrations:
2500 mg/kg/d
Basis:
nominal conc.

No. of animals per sex per dose:

1 to 17 dam(s)

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

maternal peripheral and fetal blood

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose of methanol was based on previous work [Sakanashi et al., 1994, Teratology].

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Maternal vein blood (2 to 3 µL) was collected on precleaned microscope slides 48 hours (still during pregnancy) after the final methanol treatment.
Fetal blood was taken on gestation day 18.

DETAILS OF SLIDE PREPARATION:
Slides were air dried and fixed in absolute methanol on the same day. Fixed slides were stained with 0.1 % acridine orange for 5 min, followed by a 17 min rinse in Sorensen´s M/15 phosphate buffer, pH 6.8. Slides were then wet mounted with cover slips and examined by fluorescent microscopy using a mercury lamp.

METHOD OF ANALYSIS:
Micronuclei fluoresce green-yellow. The frequency of MN was scored in 1000 reticulocytes.

Statistics:

For all statistical the pregnant dams and their litters were considered the units for comparison. Continuous variables were analysed using the two-way analysis of variance procedure and the Fisher PLSD for multiple comparisons of means. These analyses were carried out on STATVIEW SE+ (Abacus, Berkeley, CA). Incidences of frequency of micronuclei, bases on affected litters, were analyses using binomial statistics.

Key result

Sex:

female

Genotoxicity:

negative

Remarks:

The frequency of micronuclei in maternal and fetal reticulocytes was not influenced by either the marginal folic acid diet (400 nmol/kg) or by methanol treatment.

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

The frequency of MN in maternal and fetal reticulocytes was similar among the groups. The MN rate was not associated with either maternal or fetal hepatic folate concentrations. There was no significant difference in the frequency of MN in reticulocytes of fetuses that were affected by malformations compared to fetuses without malformations.

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report. No restrictions, fully adequate for assessment.

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

other: CD (Sprague-Dawley)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Manston Rd, Margate, Kent, UK
- Age at study initiation: 7-8 wks at randomisation
- Weight at study initiation: 227g (males), 184 g (females)
- Age at start of exposures: 10-11 wks
- Housing: 5/cage (same sex) in suspended stainless steel cages with wire mesh front, back and floor and stainless steel sheet sides
- Diet: SDS Rat & Mouse no. 1 SQC modified maintenance diet, Special Diet Services, Witham, Essex, UK. ad libitum (except during exposure)
- Water: Tap water provided in polypropylene bottles ad libitum (except during exposure)
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 17-23 ºC
- Humidity: 40-65 %
- Air changes (per hr): No details. Cages for each test group kept in separate ventilated cabinets
- Photoperiod: 12 hrs dark / 12 hrs light (07:30-19:30)

IN-LIFE DATES: From: 12 July 1995 To: 10 November 1995

Route of administration:

inhalation: vapour

Vehicle:

- Vehicle(s)/solvent(s) used: none

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: chambers were of stainless steel and glass construction, internal volume of 2.43m3, consisting of a cuboidal main body fitted with pyramidal base and top
- Method of holding animals in test chamber: suspended wire-mesh baskets, each with capacity of hold 10 rats, individually with a stainless steel cover over each basket. 4 such baskets were suspended in the middle portion of the exposure chamber
- System of generating vapour: test substance was metered onto a glass frit contained in a glass vessel and air was passed through at a flow rate of 80 L/min. To facilitate vapourisation, the air to the vapouriser was passed through a copper coil maintained in a water bath at 59-61ºC. The vapour/air mixture then passed into the chamber inlet ducting, where diluting air was added to achieve the appropriate vapour concentrations
- Temperature, humidity, pressure in air chamber: study means; 20.4-20.8 ºC, 56.1-66.7%
- Air flow rate: approximately 650 L/min
- Treatment of exhaust air: chamber extract

TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatography
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

5 days per week for 13 weeks

Frequency of treatment:

6 hr per day

Post exposure period:

None

Remarks:

Doses / Concentrations:
0, 1, 5, 20 ppm
Basis:
other: target concentration

Remarks:

Doses / Concentrations:
0.98, 4.96, 20.0 ppm
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
1.23, 6.5, 26.3 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

10/sex in air control and acetic anhydride exposure groups. 5/sex in positive control group (see Table 1).

Control animals:

yes, sham-exposed

Positive control(s):

cyclophosphamide;
- Justification for choice of positive control(s): cyclophosamide is known to produce highly significant increases in the frequency of micronucleated immature erythrocytes together with decreases in the proportion of immature erythrocytes
- Route of administration: oral (gastric intubation of a standard dose volume of 10 mL/kg)
- Doses / concentrations: 40 mg/kg cyclophosphamide

Tissues and cell types examined:

Bone marrow smears were prepared, stained and examined by light microscopy. The number of micronucleated cells per 1000 immature erythrocytes was determined for each animal and the proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during this assessment was also kept.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose levels of acetic anhydride (1, 5 & 20 ppm) were those selected for the 13 week inhalation repeat toxicity study based on the results of a 2 week preliminary inhalation study.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Bone marrow samples were taken from 10 male & 10 female rats in each main study group 24 hours after completion of the final exposure. For the positive control group, samples were taken from 5 males and 5 females 24 hours after dosing with cyclophosphamide.

DETAILS OF SLIDE PREPARATION:
One femur was dissected from each animal, cleaned of tissue and the contents eluted in 10 mL Hank's balanced salt solution by aspiration using a needle and syringe. The cell suspension was spun at 1000 rpm for 5 minutes. Each cell pellet was then resuspended in 2 mL filtered foetal calf serum before being sedimented out by centrifuge. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing on a glass microscope slide. Slides were stained using a modified Feulgen staining method. Several slides were prepared from each animal but only 1 slide per animal from the negative control, the high dose group and the positive control groups was examined.

METHOD OF ANALYSIS:
The number of micronucleated cells per 1000 immature erythrocytes was determined for each animal and the proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during this assessment was also kept.

Evaluation criteria:

A positive response is indicated by a substantial, statistically significant increase (p <0.01) in the incidence of micronucleated immature erythrocytes compared to the incidence for the concurrent vehicle control group; individual and/or group mean values should exceed the laboratory historical control range.
A negative result is indicated where individual and group mean incidence of micronucleated immature erythrocytes for animals treated with the test substance are not significantly greater that the incidence for the concurrent control group and where these values fall within the historical control range.
An equivocal response is obtained when the results cannot be adequately classified using the criteria for a positive or negative response.

Statistics:

Non-parametric statistical methods, based on rank, were chosen for the analysis of results. Unless there was a substantial difference in response between sexes, results for the two sexes were combined to facilitate interpretation and maximise the power of statistical analysis.
For a comparison of an individual treated group with a concurrent control group, Wilcoxon's sum of ranks test was used.

Sex:

male/female

Genotoxicity:

negative

Remarks:

see Table 2

Toxicity:

yes

Remarks:

noisy breathing, reduced weight gain and reduced food consumption in animals exposed to 20 ppm, during 13 week exposure period.

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Clinical signs were observed during the 13 week exposure period in animals exposed to 20 ppm acetic anhydride. These included partially closed eyes (observed during the first two exposures in high dose animals) and noisy breathing (with occasional instances of red staining around the snout) observed throughout most of the 13 week exposure period in high dose animals. Food consumption and weight gain was also reduced throughout the exposure period in these animals. All these signs resolved during the 13 week withdrawal period.

Table 2 Group totals/mean and results of statistical analysis

Treatment	Exposure Level	Ie / ie+me (mean %)^a	Incidence mie (mean)^a	Incidence mme (total)
Negative control	-	44	1.9	0.6
Acetic anhydride	20 ppm	41 ns	1.5 ns	0.6
Cyclophosphamide	40 mg/kg	18 **	14.1 **	0.0

ie / ie+me % - percentage of immature erythrocytes

mie - number of micronucleated cells observed in 1000 immature erythrocytes examined.

mme - number of micronucleated cells observed in 1000 mature erythrocytes examined

^a - results of statistical analysis using Wilcoxon’s sum of ranks test:

ns - not significant - p> 0.01 {1 sided probabilities},  * - p< 0.01,  ** - p< 0.001}

Table 3 Historical control values

Cummulative total of results for vehicle control animals used in previous, unrelated experiments between September 1990 to November 1993:

Cummulative results for 90 individual animals:

mie	0	1	2	3	4	5	6	7	>7
Frequency	36	33	16	4	1	0	0	0	0

mie  The incidence of micronucleated cells per 1000 immature erythrocytes

Frequency - The number of times that result has been obtained

Conclusions:

Acetic anhydride did not cause chromosomal damage in rat bone marrow following inhalation exposure at 20 ppm.

Executive summary:

Since the test substance did not cause any substantial increase in the incidence of micronucleated immature erythrocytes or any substantial decrease in the proportion of immature erythrocytes, it is concluded that acetic anhydride did not show any evidence of causing chromosome damage or bone marrow cell toxicity following subchronic inhalation exposure of rats in this in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

There are several data available on the registered substance itself as well as suitable read-across substances (hydrolysis products methanol and acetic acid), all assessed with at least Klimisch 2, sufficiently covering all required endpoints, i.e. gene mutation in both bacteria and mammalian cells, and chromosome aberrations (micronucleus induction) in mammalian cells. Those in vitro genetic toxicity tests are designed, i.a. via addition of additional enzymes (metabolic activation; Aroclor 1254 induced rat liver S9), to mimic the exposure of humans to a potential genotoxic agent very close. They are accepted surrogates for human data, and no indication is given that the obtained results are not relevant for humans / risk assessment. Further, in vivo data similarly indicate no genotxic potential. Hence, the database is of good quality, sufficient to exclude that any risk with regard to genotoxic effects may arise for humans from TMOA.

All available tests gave consistently negative results, which makes a mode of action analysis impossible, as no conclusions from any events can be drawn.

Justification for classification or non-classification

All available in vitro studies on TMOA and suitable read-across substances (hydrolysis products methanol and acetic acid) covering the endpoints gene mutation in bacteria and mammalian cells and micronucleus induction in various cell systems and even in rodents in vivo (micronucleus assay) gave negative results or were shown to pose no relevant potential in humans. All these stated above cover the main causes for genetic damage sufficiently and mimic due to the addition of S9 in vivo metabolism to a wide extent, and are partially even in vivo data. Based on the available data, there is no indication given that TMOA should be classified as mutagen.