Maleic anhydride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The functioning of all compartments of mammalian organisms at the cellular level is based on processes in physiological aqueous medium. Upon contact with water, the target substance maleic anhydride hydrolyses rapidly and completely (DT50 = 0.3 min) to form maleic acid. Therefore, physiological processes on a cellular or compartmental level will react to the product of hydrolysis, maleic acid, rather than the short-lived maleic anhydride. Consequently, a read-across approach using maleic acid is justified to investigate toxicological effects of maleic anhydride on mammalian organisms. Thus, the genotoxic potential of the target substance maleic anhydride is assessed in a weight-of-evidence approach based on several in vitro studies (OECD 471, OECD 476) with the target substance itself and maleic acid.

In conclusion, these studies are considered negative with respect to genotoxicity. There were not reliable in vitro cytogenicity assays on maleic anhydride or maleic acid available. Therefore, in combination with a negative in vivo bone marrow chromosome aberration test (OECD 475) with maleic anhydride, it is concluded that maleic anhydride is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 strains tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction from Aroclor 1254 pretreated male SD rats and Syrian hamsters with NADPH-generating system
Type and composition of metabolic activation system:
- source of S9 : Male Sprague-Dawley rats and Syrian hamsters
- method of preparation of S9 mix : Liver S-9 fractions were routinely prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected, ip, with Aroclor 1254 (200 mg/ml in corn oil) at 500 mg/kg . Five days after injection, the aninials were sacrificed by decapitation (EGG, SRI) or cervical dislocation (CWR) and the livers were removed aseptically . The aninials were fasted for 12-24 hr immediately preceding sacrifice. Liver homo-enates were prepared aseptically at 0-4°C. Excised livers were rinsed with 0.15 M KCI, then minced and homogenized (3 mL of 0.15 M KCI/g wet tissue) in a Potter-Elvehjem apparatus with a teflon pestle (EGG, SRI) or in a Waring blender (CWR). The homogenate was centrifuged for 10 min at 9000 g at 4°C. The supernatant (S-9) was decanted and distributed into freezing ampules and stored at -70°C.
- concentration or volume of S9 mix and S9 in the final culture medium: One milliliter of S-9 mix has the following composition : S-9 . 0.10 mL ; 0.04 M MgCI, 0.02 mL; 1.65 M KCI, 0.02 mL; 0.04 M ß-nicotinamide adenine dinucleotide phosphate (NADP), 0.10 mL; 0.05 M glucose-6-phosphate; 0.10 mL; 1.0 M NaH2PO4 (pH 7.4), 0.10 mL; and distilled water, 0 .56 mL.

Test concentrations with justification for top dose:

0.0, 1.0, 3.3, 10.0, 33.3, 100.0, 333.3, 1000.0 or 3333.3 µg/plate

Vehicle / solvent:

- Vehicle: acetone

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

all strains in the presence of rat and hamster S-9

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamin

Remarks:

TA98 without S-9

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100 and TA 1535 without S-9

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S-9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration triplicate unless samples marked toxic or contaminated were excluded from mean and SEM calculations

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period :20 min at 37°C
- Exposure duration/duration of treatment : for 48 h at 37°C

Evaluation criteria:

A positive response was indicated by a reproducible, dose-relatedincrease, whether it be twofold over background or not.
A "questionable"(or "inconclusive") response applied to low-level responses that were not reproducible
within the laboratory or to results that showed a definite trend but with whichthe investigator did not feel comfortable in making a"positive" or "negative" decision. It also included tests in which an elevated revertant colony yield occurred at only a single dose level.

Statistics:

yes, no further details provided.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>/= 100 µg/plate (with and without S9 mix from rat livers)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>/= 100 µg/plate (with and without S9 mix from rat livers)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>/= 100 µg/plate (with and without S9 mix from rat livers)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>/= 100 µg/plate (with and without S9 mix from rat livers)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

Under the experimental conditions reported, maleic anhydride did not cause gene mutations in an Ames Test (equivalent to OECD 471). Therefore, the test item is considered to be non-mutagenic in this bacterial reverse gene mutation assay.

Executive summary:

In a bacterial reverse gene mutation assay (equivalent to OECD guideline 471), strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium were exposed to maleic anhydride in acetone at concentrations of 1.0, 3.3, 10.0, 33.3, 100.0, 333.3, 1000.0 or 3333.3 μg/plate in the presence and absence of mammalian metabolic activation (liver S-9 fractions from male rats and hamsters) . The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains and two experiments. Furthermore, there were signs of toxicity observed in all strains tested at or above 100.0 µg/plate with or without S-9 from rats. Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

The functioning of all compartments of mammalian organisms at the cellular level is based on processes in physiological aqueous medium. Upon contact with water, maleic anhydride hydrolyses rapidly and completely (DT50 = 0.3 min) to form maleic acid (Bunton et al., 1963). Therefore, physiological processes on a cellular or compartmental level will react to the product of hydrolysis, maleic acid, rather than the short-lived maleic anhydride. Consequently, a read-across approach using maleic acid is justified to investigate toxicological effects of maleic anhydride on mammalian organisms.

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. No precipitation was observed by the unaided eye up to the maximum concentration.

Conclusions:

In this study, under the given conditions, the results of the mutagenic potential of the test item maleic acid, were found to be negative.

Executive summary:

In a mammalian cell HPRT gene mutation assay conducted in accordance with OECD 476, V79 cells cultured in vitro were exposed to maleic acid (purity 99.5%) in 10% v/v deionised water investigated at concentrations of 37.5; 75.0; 150; 300; 600; and 1200 µg/mL in the presence and absence of mammalian metabolic activation (experiment I, 4-hour exposure). For experiment II (24 hour exposure), concentrations tested were 75.0; 150; 300; 600; 900; and 1200 µg/mL in the presence and absence of metabolic activation. 7,12- dimethylbenzanthracene (DMBA) and ethylmethanesulphonate (EMS) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency, showing the validity of the experiments.

No precipitation of the test item was observed up to the maximal concentration in all experiments. Cytotoxic effects as indicated by a relative cloning efficiency I of less than 50 % in both parallel cultures solely occurred at 900 μg/mL and above in experiment II without metabolic activation following 24 hours of exposure. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of the second experiment without metabolic activation at 900 and 1200 μg/mL. This effect, however, was not reproduced in the parallel culture performed under identical conditions. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 statistics software. A significant trend of the mutation frequency was solely determined in the first culture of the first experiment without metabolic activation. This trend was judged as irrelevant fluctuation however, since it actually was reciprocal, going down versus increasing concentrations.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Maleic acid is considered to be non-mutagenic in this HPRT assay.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 476 for In vitro Mammalian Cell Gene Mutation Test data.

This information is used in a read-across approach in the assessment of the target substance. The functioning of all compartments of mammalian organisms at the cellular level is based on processes in physiological aqueous medium. Upon contact with water, maleic anhydride hydrolyses rapidly and completely (DT50 = 0.3 min) to form maleic acid. Therefore, physiological processes on a cellular or compartmental level will react to the product of hydrolysis, maleic acid, rather than the short-lived maleic anhydride. Consequently, a read-across approach using maleic acid is justified to investigate toxicological effects of maleic anhydride on mammalian organisms.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Maleic anhydride was tested negative in an in vivo bone marrow chromosome aberration assay (equivalent to OECD 475) and cited in maleic anhydride OECD SIDS (2004) report.

Link to relevant study records

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983-03-14 to 1983-06-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

(positive controls were not used, only 50 cells per animal were analyzed)

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Remarks:

albino

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Housing: individually
- Diet: ad libitum (except during the exposure period)
- Water: ad libitum (except during the exposure period)
- Acclimation period: 19 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-27
- Humidity (%): 32-66
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation

Vehicle:

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

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Atmospheres were generated by heating maleic anhydride in flasks and transporting the vapors with nitrogen gas to 5 m³ glass and stainless steel chambers.
- Temperature, humidity in air chamber: 23-25°C, 36-45%
- Air flow rate: 1 L/min


TEST ATMOSPHERE
- Brief description of analytical method used: Atmospheric concentrations were monitored five times during the exposure period with two methods: 1. chamber samples were bubbled through distilled water and maleic acid was determined by a HPLC method; 2. chamber samples were drawn through a glass tube filled with p-Anisidine coated with XAD-resin beads, and the maleic anhydride derivative was determined by HPLC (methods provide a measure of total maleic (maleic acid plus maleic anhydride converted to maleic acid) and maleic anhydride).
- Samples taken from breathing zone: yes (5 times in 6 hours)

Duration of treatment / exposure:

6 hrs

Frequency of treatment:

once

Post exposure period:

Sacrifice at 6, 24, and 48 hours.

Dose / conc.:

0 mg/m³ air (nominal)

Dose / conc.:

1 mg/m³ air (nominal)

Remarks:

analytical concentration range: 1.66 - 3.06 mg/m³; average concentration of 2.34 mg/m³.

Dose / conc.:

100 mg/m³ air (nominal)

Remarks:

analytical concentration range: 20.43 - 49.29 mg/m³; average concentration of 33.30 mg/m³.

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

Positive control(s):

none

Tissues and cell types examined:

Bone marrow cells were collected and processed for analysis.

Details of tissue and slide preparation:

SAMPLING TIMES: Five animals per sex per group were killed 6, 24 or 48 hours following termination of exposure.


DETAILS OF SLIDE PREPARATION: Bone marrow cells were removed by aspiration from both femurs of rat immediately after sacrifice. Subsequently, the cells were fixed, dropped on slides and stained. Two slides were prepared for each animal and analyzed by an individual who was unfamiliar with the animals identity. Generally, fifty cells in metaphase were examined from each rat that provided analyzable cells.


METHOD OF ANALYSIS: The following parameters were calculated for each animal: numbers and type of chromosomal aberrations, mitotic index, chromosome number, and vernier location each metaphase containing damage .

Statistics:

The mean mitotic indices, mean modal numbers, percent aberrant cells and the total number of aberrations per animal for each group were statistically compared using the Kruskal-Wallis nonparametric analysis of variance and nonparametric all pairwise group comparison.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

No deaths occurred during the study but all treated animals were sluggish during exposure and many of the high-dose group had squinted eyes and bloody encrusted nose. One hour after exposure, all rats in the high dose group appeared sluggish and there was a bloody crust around the nose of four males and six females. No evidence of toxicity in the bone marrow cells were observed. There were no treatment-related effects on the frequency of chromosomal aberrations at any of the times examined. A statistically significant increase (p=0.022) in chromosome number was observed in the low-dose animals at 6 hours and in the high-dose animals at 24 hours (p=0.045); these were not considered treatment-related. None of the rats had polyploid or aneuploid cells and none had consistently abnormal chromosome numbers.

Conclusions:

In this study, under the given conditions, maleic anhydride is not considered to be clastogenic at any of the dose levels tested.

Executive summary:

In a Sprague-Dawley albino rats bone marrow chromosomal aberration assay (equivalent to OECD Guideline 475), 15 rats/sex/concentration were treated via inhalation route with maleic anhydride (100 % purity). at concentrations of 0, 1 or 100 mg/m³. Bone marrow cells were harvested at 6, 24- and 48-hours post-treatment.

There were signs of toxicity during the study. All treated animals were sluggish during exposure and many of the high-dose group had squinted eyes and bloody encrusted nose. One hour after exposure, all rats in the high dose group appeared sluggish and there was a bloody crust around the nose of four males and six females. However, no evidence of toxicity in the bone marrow cells were observed. There were no treatment-related effects on the frequency of chromosomal aberrations at any of the times examined. A statistically significant increase (p=0.022) in chromosome number was observed in the low-dose animals at 6 hours and in the high-dose animals at 24 hours (p=0.045); these were not considered treatment-related. None of the rats had polyploid or aneuploid cells and none had consistently abnormal chromosome numbers.

This study is classified as acceptable with some restrictions ( purity of the test substance was not known and positive controls were not used) and satisfies the requirement for Test Guideline OECD 475 for in vivo cytogenetic mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The functioning of all compartments of mammalian organisms at the cellular level is based on processes in physiological aqueous medium. Upon contact with water, the target substance maleic anhydride hydrolyses rapidly and completely (DT50 = 0.3 min) to form maleic acid. Therefore, physiological processes on a cellular or compartmental level will react to the product of hydrolysis, maleic acid, rather than the short-lived maleic anhydride. Consequently, a read-across approach using maleic acid is justified to investigate toxicological effects of maleic anhydride on mammalian organisms. Thus, the assessment of the genotoxic potential can be based on available data on maleic acid.

The target substance itself was tested negative in a bacterial reverse mutation test (OECD 471). The read-across partner maleic acid was tested negative in an in vitro HPRT assay (OECD 476). Maleic anhydride was tested negative in an in vivo bone marrow chromosome aberration assay (equivalent to OECD 475).

Maleic anhydride was not mutagenic in a preincubation Ames test with and without metabolic activation (tested up to 3333.3 µg/plate in Salmonella typhimurium TA1535, TA1537, TA98, and TA100; metabolic activation: liver S-9 mix from Aroclor 1254-induced male Sprague-Dawley rats and Syrian hamsters). Cytotoxicity (reduction of the background lawn) was observed >/= 100 µg/plate (depending on the strain and the activation system; no cytotoxicity was observed with S-9 mix from hamster liver). This finding was supported by Ames tests with reduced S. typhimurium tester strain battery, maleic anhydride was also not mutagenic with and without metabolic activation.

The potential of maleic acid to induce gene mutations was investigated at the HPRT locus in V79 cells of the Chinese hamster in an OECD 476 guideline study. The highest applied concentration (1200 µg/mL) was equal to a molar concentration of approximately 10 mM. The test substance did not induce gene mutations at the HPRT locus in V79 cells neither with nor without metabolic activation.

The negative results in the in vitro genetic toxicity studies were supported by a reliable in vivo bone marrow chromosomal aberrations (cited in OECD SIDS for CAS. Nos. 108-31-8/ 110-16-7, 2004) with maleic anhydride. The chromosomal aberration test was equivalent to the OECD Guideline 475 with acceptable restrictions. The route of administration was inhalation, the concentrations administered were up to 33 ppm (analytical concentration.) The test gave negative results.

Therefore, based on available data from the target substance itself and from its suitable read-across partner, the target substance maleic anhydride is considered negative for mutagenicity based on a weight-of-evidence approach.

Justification for classification or non-classification

Based on available data from the target substance itself and also from its read-across partner, the target substance maleic anhydride is considered negative for mutagenicity.