Acetic anhydride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Acetic anhydride has been examined for mutagenicity in vitro in recognised core assays. It has shown negative results for bacterial mutagenicity in vitro, and although the formal evaluation of the in vitro mouse lymphoma mammalian cell gene mutation assay is inconclusive, there is no evidence from the data for acetic anhydride and its hydrolysis product, acetic acid, for likely significant mutagenicity in mammalian cells. It is concluded that the available data indicate that acetic anhydride has no significant genotoxicity in vitro. This finding is supported by the negative result from the in vivo bone marrow micronucleus assay. It is recognised that since acetic anhydride is a reactive molecule, this assay involving the bone marrow will not be stringent for possible local effects. However, there are no data from the in vitro assays to indicate that any significant local mutagenic activity from acetic anhydride would be expected, and it is generally considered that there is a lack of clear evidence for anhydrides as a class (HSE, 2002). The available data from in vitro studies suggest that at excessive and unphysiological concentrations of acetic anhydride, the pH of the test system is significantly reduced and inconclusive results can be induced as a consequence of this. However, these results are at doses exceeding the limit dose for the relevant assays, which cause circumstances that are now accepted in the literature as being artificial and of no value for contributing to an evaluation of the genotoxicity of a chemical (EU DAR, 2008). There are limited data indicating that this may also be the case in vivo. It is concluded that the available data indicate that acetic anhydride has no significant genotoxic activity.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key 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other: GLP compliant, guideline study, available as unpublished report.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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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 ploypropylene bottles ad libitum (except during exposure).
- Acclimation period: 4 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 ºC
- Humidity (%): 40-65 %
- Air changes (per hr): No details. Cages for each test group kept in separate ventilated cabinets.
- Photoperiod (hrs dark / hrs light): 12/12 (0730-1930)

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 - 61+ ºC. The vapour/air mixture then passed into the chamber inlet ducting, where diluting air was added to achieve the appropriate vapoour concnetrations.
- 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 h per day

Post exposure period:

None

Remarks:

Doses / Concentrations:
0, 1, 5, 20 ppm
Basis:
other: Target concentrations

Remarks:

Doses / Concentrations:
0, 4.18, 20.9, 83.5 mg/m3
Basis:
other: Target concentrations

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 icronucleated immature erythrocytes together with decreases in the proportion of immature erythrocytes.
- Route of administration: oral (gastric intubation of a standard dose volume of 10ml/kg).
- Doses / concentrations: 40 mg/kg.

Tissues and cell types examined:

Bone marrow smears were prepared, stained and examined by light microscopy. The number of micronucleated cells per 1000 immature erthrocytes 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 erthrocytes 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

Toxicity:

yes

Remarks:

Noisy breathing, reduced weight gain and reduced food consumption in animals exposed to 20ppm, 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 expsoures 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.

Histological changes in the respiratory tract after 13 week exposure to acetic anhydride were dose related and consistent with changes typical of a substance acting as a local/contact irritant . These changes were predominantly localised inflammatory lesions with subsequent areas of epithelial hyperplasia and/or squamous metaplasia and regressed during the withdrawal period, indicating that long-term damage had not been caused within the respiratory tract. 1 ppm was a pathological no-effect level.

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    }

                  *        -       p< 0.01    } 1 sided probabilities

                                      **      -       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:

Interpretation of results (migrated information): negative
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)

Additional information

Additional information from genetic toxicity in vivo:

Non-human information

In vitro data

The key studies are considered to be bacterial mutation assays (Mortelmans et al., 1986, McMahon et al., 1979) and a mammalian cell gene mutation assay (Seifried et al 2006, HSE 2002). These are two recognised core assay types for investigating mutation in vitro. Considering in vitro mammalian cell cytogenetics, there is not a study on acetic anhydride itself. However acetic anhydride hydrolyses rapidly in aqueous media to release 2 moles of acetic acid per mole of acetic anhydride and so data on acetic acid is considered to meet this endpoint requirement (Morita et al., 1990). 

Acetic anhydride was tested in a standard Ames test (Mortelmans et al., 1986). Salmonella typhimurium (strains TA1535, TA1537, TA98 and TA100) was treated with acetic anhydride both with and without metabolic activation (S9). A range of doses was used up to 10,000 μg/plate where toxicity allowed. In another evaluation using a modified Ames test, acetic anhydride was tested in these same strains, plus additional bacterial strains, including S typhimurium TA1538, E coli WP2 and E coli WP2uvrA-. Acetic anhydride was negative, ie non-mutagenic, in both of these studies. Other evaluations in bacterial genotoxicity assays have given confirmatory negative results (HSE, 2002).

The data indicate that acetic acid is not mutagenic in bacterial test systems.

As considered above, although no relevant study is available for acetic anhydride, acetic acid has been examined for cytogenetic activity in the absence and presence of S9 in Chinese hamster ovary K1 cells (Morita et al., 1990). The evaluation included an examination of the relationship between the pH of the medium and clastogenic activity.  In CHO cells exposed to acetic acid a dose-dependent increase in chromosomal aberrations was observed beginning at a concentration of 10 mM, in the absence of S9, and at 8 mM, in the presence of S9. These concentrations were close to the cytotoxic limit at which cells could no longer be evaluated. This activity was removed when the pH of the medium was maintained near neutral through the use of media with increased buffering capacity, or by neutralisation with sodium hydroxide. This activity, observed at or near the limit dose for this assay type (10 mM), and which is removed when the pH is maintained at near neutral, is considered to be a function of the pH change induced by acetic acid at very high concentrations, and not due to genotoxicity of the chemical itself. This is supported by negative findings in other cytogenetic assays using standard dose levels, or positive results only when the pH is once again reduced through excessively high concentrations of acetic acid (EU DAR, 2008).

It is concluded that acetic acid and therefore acetic anhydride has no significant clastogenic activity in vitro.

Acetic anhydride has been examined for mutagenic activity in mammalian cells in vitro using the mouse lymphoma L5178Y assay (Seifried et al., 2006: HSE, 2002). In the presence of S9, no increase in mutant frequency was observed. In the absence of S9, an increase in mutant frequency was observed, but only at a dose level inducing excessive toxicity (6% relative total growth, against a limit of 10%). The assay was repeated, but again three out of four dose levels produced excessive toxicity (6% relative total growth or less). The remaining dose level produced toxicity of 21% relative total growth, with just a two-fold increase in mutant frequency over controls. The observed increase in mutant frequency was small and appears to be associated with the increasing cytotoxicity induced by the treatment. 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. A recent evaluation of these data has reported them as inconclusive (Seifried et al., 2006). 

Considering this further,in the cytogenetic assays for acetic anhydride and acetic acid (chromosomal damage and sister chromatid exchange discussed above), effects were only seen at very high doses (non-physiological doses and ones exceeding the guideline limit doses for the assays) and these were due to the induced pH changes in the cell culture systems. These data indicate that were acetic anhydride to be examined in an in vitro mammalian chromosome aberration testsuch as the Chinese hamster ovary assay, it would show no genotoxic activity at standard dose levels, but similar to the cytogenetic assays and at excessively high doses, the pH of the cell culture system would be altered, and effects may be seen. However, any such effects would be of no relevance for any meaningful hazard assessment of acetic anhydride. This phenomenon of irrelevant effects at reduced pH levels in mammalian cell gene mutation assays has already been reported (Cifone et al., 1987) [see comment in table above], including reference to the effect being induced by several short chained aliphatic acids.

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

In vivo data

The key study is considered to be an in vivo cytogenetic study (bone marrow micronucleus) in the rat (HRC, 1996a).  Acetic anhydride was examined in a bone marrow micronucleus assay in the rat, following exposure of animals for 6h/day and 5 days/week for 13 weeks to concentrations of 20 ppm (83.5 mg/m3). Bone marrow was sampled from each of 10 males and 10 females at 24h after the last exposure. No significant increase in the micronucleus frequency in immature erythrocytes was observed. In addition, no significant decrease in the proportion of immature erythrocytes in the total erythrocyte population was observed, suggesting that acetic anhydride was not toxic to the bone marrow under the conditions of exposure. 

Human information

There is no information indicating any adverse effects of acetic anhydride.

 

Justification for selection of genetic toxicity endpoint
This study is an in vivo investigation of genotoxicity and is the only study which is reliable without restriction (Klimisch score=1). There are a series of in vitro studies which are negative or inconclusive and are reliable with restrictions or unassigned (Klimisch score=2 or 4).

Justification for classification or non-classification

It is concluded that acetic anhydride has no significant genotoxic activity and therefore does not warrant classification.