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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1980-01-01 to 1990-01-31
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990
Reference Type:
publication
Title:
Unnamed
Year:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The methods followed those first described by Ames et al. (1975) and were consistent with subsequently adopted international test guidelines. Succinic anhydride was not tested for mutagenicity using strain TA 102. Succinic anhydride was tested in two laboratories for induction of gene mutations in several strains of Salmonella typhimurium by a preincubation protocol with and without Aroclor 1254-induced male Sprague Dawley rat or Syrian hamster liver S9 (Zeiger et al., 1987); no mutagenic activity was observed in any of the strains (TA97, TA98, TA100, TA1535, or TA1537).
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Succinic anhydride
EC Number:
203-570-0
EC Name:
Succinic anhydride
Cas Number:
108-30-5
Molecular formula:
C4H4O3
IUPAC Name:
succinic anhydride
Specific details on test material used for the study:
- Name of the test material used in the report: Succinic anhydride
- Source: Aldrich
- Purity: 99.9%

Method

Target gene:
histidine-dependence
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254-induced male Sprague Dawley rat or Syrian hamster liver
Test concentrations with justification for top dose:
0, 3, 10, 33, 100, 333, 666, 1000, 3333, and 10000 ug succinic anhydride/plate. The high dose was limited by toxicity or solubility but did not exceed 10 mg/plate.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: with metabolic activation (+S9): all strains: 2-aminoanthracene; -S9: TA98: 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
Succinic anhydride was incubated with Salmonella typhimurium tester strains TA97, TA98, TA100, TA1535, and TA1537 either in buffer or S9 mix (metabolic activation enzymes and cofactors from Arochlor 1254-induced male Sprague Dawley rat or Syrian hamster liver) for 20 minutes at 37° C before the addition of soft agar supplemented with L-histidine and D-biotin and subsequent plating on minimal glucose agar plates. Incubation was continued for an additional 48 hours. Succinic anhydride was tested in a series (four strains used) in each of two different laboratories. If all results were negative, the chemical was retested in all strains. In the second laboratory, repeat trails were performed with a different concentration of S9. Each test consisted of triplicate plates of concurrent positive and negative controls and of at least five doses of the study chemical. The high dose was limited by toxicity or solubility but did not exceed 10 mg/plate.
Evaluation criteria:
A positive response was defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response was defined as an increase in revertant which was not dose related, not reproducible, or of insufficient magnitude to support a determination of mutagenicity. A response was considered negative when no increase in revertant colonies was observed after chemical treatment.
Statistics:
Not applicable

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 3333 µg/plate (+S9)/ 333 µg/plate (-S9)
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 3333 µg/plate (+S9)/ 333 µg/plate (-S9)
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 3333 µg/plate (+S9)/ 1000 µg/plate (-S9)
Vehicle 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:
cytotoxicity
Remarks:
at 3333 µg/plate (+S9)/ 333 µg/plate (-S9)
Vehicle 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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the first laboratory trial, cytotoxicity was not seen at any concentration with or without metabolic activation. In the second laboratory trial, slight toxicity was seen in the absence of S9 at concentrations of 333 ug/plate succinic anhydride and greater. In the presence of 10% and 30% S9, slight toxicity was seen at concentrations of 3333 ug/plate succinic anhydride and greater.

Any other information on results incl. tables

Table 1. Mutagenicity of succinic anhydride in Salmonella typhimurium (Laboratory 1)

                Revertants/Platea    
        - S9     + S9 (hamster)     + S9 (rat)
 Strain  Dose (ug/plate)  Trail 1  Trial 2 Trial 1  Trial 2    Trial 1  Trial 2
 TA100  0 (solvent control)  94 + 4.4 123 + 7.5  131 + 8.5  111 + 9.9  111 + 6.1  115 + 5.2 
   3 118 + 16.5  --  --   --  --  -- 
   10 --  99 + 12.3 --  --  --  -- 
   33 114 + 6.1  120 + 3.7  --  --  -- -- 
   100 101 + 1.5  103 + 6.0  124 + 8.2  93 + 4.0  129 + 1.2  110 + 7.5 
   333  113 + 6.1  93 + 9.3 116 + 11.5  104 + 6.6  127 + 7.3  120 + 5.0 
   666 87 + 12.0  41 + 2.3  --  --  --  -- 
   1000 -- --  111 + 9.6  107 + 5.0 114 + 8.3  103 + 6.4 
   3333 --  --  107 + 9.3  81 + 7.0  97 + 6.1  105 + 4.4 
   10000 --  --  105 + 16.7   71 + 8.5 99 + 9.2  77 + 9.5 
 Trial summary  Negative  Negative  Negative  Negative  Negative  Negative
   Positive controlb  431 + 13.3 411 + 28.4  1500 + 142.6  1358 + 108.2  481 + 28.2  721 + 14.5 
                      
 TA 1535  0 (solvent control)  19 + 2.5 30 + 2.2  9 + 2.0  7 + 1.2  9 + 2.3  11 + 2.7 
   3 27 + 0.6  --  --  --  --  -- 
   10 --  32 + 2.1  --  --  --  -- 
   33 20 + 0.3  25 + 3.0  --  --  --  -- 
   100 25 + 2.1  30 + 4.9  10 + 2.6  6 + 0.6  9 + 2.2  10 + 2.6 
   333  22 + 1.7 19 + 3.5  8 + 2.1  6 + 0.6  9 + 1.5  7 + 0.3 
   666 7 + 3.1  6 + 4.3  --  --  --  -- 
   1000 --  --  11 + 2.5  7 + 1.2  11 + 1.2  6 + 0.6 
   3333 --  --  6 + 1.2  6 + 1.3  5 + 0.3  9 + 0.0 
   10000 --  --  4 + 0.7  6 + 0.0  4 + 0.9  6 + 0.0 
   Trial summary  Negative  Negative  Negative  Negative  Negative  Negative
    Positive controlb  511 + 11.6 436 + 10.7  503 + 14.7  389 + 3.5  186 + 15.3  167 + 3.2 
                      
 TA 1537  0 (solvent control)  5 + 1.2 7 + 2.0  6 + 1.5  9 + 1.7  8 + 2.3  9 + 2.0 
   3 5 + 0.9  --  --  --  --  -- 
   10 --  5 + 1.0  --  --  --  -- 
   33 4 + 0.9  7 + 2.5  --  --  --  -- 
   100 6 + 1.2  5 + 1.2  8 + 2.0  10 + 2.9  9 + 2.1  5 + 0.6 
   333 5 + 1.8  5 + 2.0  8 + 0.6  7 + 0.3  11 + 1.2  6 + 1.5 
   666 3 + 0.9  2 + 0.7  --  --  --  -- 
   1000 --  --  7 + 1.7  8 + 0.0  5 + 1.5  5 + 1.2 
  3333 --  --  6 + 2.8  5 + 1.5  6 + 0.9  7 + 1.2 
  10000  --  --  5 + 2.2  6 + 1.0  8 + 2.2  7 + 0.9 
   Trial summary  Negative  Negative  Negative  Negative  Negative  Negative
    Positive controlb  248 + 86.7 166 + 31.4  462 + 22.6  321 + 25.6  147 + 16.6  179 + 19.8 
                      
 TA98 0 (solvent control)  22 + 5.0  16 + 2.1  25 + 2.3  23 + 3.3  24 + 3.2  26 + 2.0 
   3 16 + 1.5  --  --  --  --  -- 
   10 --  15 + 0.3  --  --  --  -- 
   33 19 + 3.4  14 + 0.9  --  --  --  -- 
   100 16 + 1.2  16 + 0.9   33 + 2.1 23 + 3.3  24 + 4.9  26 + 0.3 
   333 12 + 1.8  11 + 1.9  27 + 2.1  23 + 3.7  24 + 2.5  19 + 0.3 
   666 7 + 0.9  4 + 0.3  --  --  --  -- 
   1000 --  --  23 + 2.5  20 + 0.6  29 + 2.0  27 + 2.8 
   3333 --  --  18 + 0.3  22 + 4.5  25 + 5.1  23 + 3.0 
   10000 --  --  19 + 1.5  15 + 1.7  24 + 2.1  19 + 3.8 
   Trial summary  Negative  Negative  Negative  Negative  Negative  Negative
  Positive controlb   757 + 11.3 793 + 19.0  1287 + 249.6  1229 + 84.4  355 + 43.6  474 + 45.7 
aRevertants are presented as mean + standard error from three plates; bPositive control: 2 -aminoanthracene used on all strains in the presence of S9. In the absence of metabolic activation, 4 -nitro-o-phenylenediamine was used with TA98, sodium azide was used with TA100 and TA1535, and 9 -aminoacridine was used with TA1537 and TA97.             

Table 2. Mutagenicity of succinic anhydride in Salmonella typhimurium (Laboratory 2)

              Revertants/Platea    
        - S9     + S9 (hamster)     + S9 (rat)
 Strain  Dose (ug/plate)  Trail 1  Trial 2 10% 30%    10%  30%
 TA100  0 (solvent control)  100 + 4.3 128 + 9.5  94 + 8.4  118 + 6.6  96 + 1.0  103 + 2.3 
   3.3 -- 141 + 9.4  --   --  --  -- 
   10 --  124 + 8.5 --  --  --  -- 
   33 -- 126 + 15.3  --  119 + 8.4  -- 108 + 8.1
   100 88 + 8.1  130 + 3.0  90 + 1.7  106 + 7.8  100 + 4.8  108 + 4.3 
   333  74 + 3.5  112 + 10.4c 94 + 7.2  103 + 2.7  101 + 4.9  107 + 11.6 
   1000 52 + 16.5c  -- 104 + 5.2 104 + 3.8 83 + 2.6 120 + 2.1 
   3333 Toxic --  88 + 5.6c 87 + 2.3 80 + 10.5c  88 + 10.0 
   6666 74 + 0.0c --  89 + 6.2c  -- Toxic -- 
 Trial summary  Negative  Negative  Negative  Negative  Negative  Negative
   Positive controlb  1096 + 18.8 1130 + 47.3  890 + 31.9  385 + 31.2  1408 + 48.0  806 + 19.3
                      
 TA 1535  0 (solvent control)  29 + 2.0 27 + 5.9  12 + 0.3  13 + 3.3  13 + 0.3  12 + 1.8 
   3.3 29 + 0.3  25 + 4.7  --  --  --  -- 
   10 20 + 3.0 24 + 2.2  --  --  --  -- 
   33 25 + 2.5  19 + 1.8  --  10 + 0.9 --  14 + 0.9
   100 22 + 3.4  23 + 4.5  9 + 1.7  13 + 1.5  7 + 2.5  13 + 1.9 
   333  20 + 2.1c 21 + 1.2c  8 + 2.4  16 + 1.7  8 + 1.3  13 + 0.6 
   1000 --  --  9 + 1.5  15 + 0.9  12 + 2.3  11 + 0.6 
   3333 --  --  8 + 0.9c  11 + 2.0  9 + 2.7c  11 + 1.8 
   6666 --  --  5 + 0.9c  -- Toxic  -- 
   Trial summary  Negative  Negative  Negative  Negative  Negative  Negative
    Positive controlb  849 + 29.3 53 + 2.2  61 + 3.2  91 + 4.0  85 + 1.0  74 + 0.6 
                      
 TA 97  0 (solvent control)  90 + 2.5 98 + 11.3  101 + 2.7  118 + 4.9  92 + 4.3  188 + 6.8 
   3.3 92 + 3.1  106 + 3.0 --  --  --  -- 
   10 91 + 3.5  90 + 3.7  --  --  --  -- 
   33 86 + 7.4  100 + 3.7  --  118 + 5.3  --  185 + 6.9 
   100 88 + 4.6  89 + 9.6  111 + 1.2  155 + 9.9  88 + 10.7  184 + 5.8 
   333 79 + 4.7c  77 + 5.6c  97 + 10.1  143 + 11.6  72 + 7.5  185 + 8.8 
   1000 --  --  99 + 7.4  131 + 4.5  106 + 1.8  186 + 9.4 
  3333 --  --  80 + 2.0c  115 + 1.2  82 + 1.0c  97 + 6.4 
  6666  --  --  Toxic -- 49 + 6.6c  --
   Trial summary  Negative  Negative  Negative  Negative  Negative  Negative
    Positive controlb  611 + 67.2 414 + 22.9  538 + 8.4  317 + 15.6  883 + 43.6  438 + 11.3 
                      
 TA98 0 (solvent control)  19 + 1.9  17 + 1.5  29 + 5.4  26 + 3.6  29 + 1.5  38 + 4.8 
   3.3 15 + 1.2  18 + 3.8  --  --  --  -- 
   10 14 + 3.2 23 + 5.2  --  --  --  -- 
   33 18 + 2.6  25 + 2.3  --  23 + 2.7 --  29 + 4.0 
   100 20 + 1.7  18 + 1.5   25 + 2.0 28 + 2.1  24 + 0.7  37 + 3.2 
   333 12 + 3.0c  16 + 4.9  21 + 3.4  31 + 3.6  28 + 3.8  30 + 1.3 
   1000 --  --  28 + 3.0  29 + 4.1  24 + 4.9  32 + 4.5 
   3333 --  --  23 + 3.4c  30 + 2.6  14 + 1.0c  24 + 1.3c 
   6666 --  --  46 + 37.5c  -- 7 + 0.3c  --
   Trial summary  Negative  Negative  Negative  Negative  Negative  Negative
  Positive controlb   168 + 8.9 170 + 17.2  480 + 20.3  80 + 5.8  993 + 26.8  202 + 11.9 
aRevertants are presented as mean + standard error from three plates; bPositive control: 2 -aminoanthracene used on all strains in the presence of S9. In the absence of metabolic activation, 4 -nitro-o-phenylenediamine was used with TA98, sodium azide was used with TA100 and TA1535, and 9 -aminoacridine was used with TA1537 and TA97; cSlight toxicity             

Applicant's summary and conclusion

Conclusions:
No mutagenic response was observed when succinic anhydride was tested in the Ames assay up to 10 mg/plate which exceeds the curent limit concentration recommended by OECD TG 471.
Executive summary:

In a reverse gene mutation assay in bacteria performed similar to OECD 471, TA 97, TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium were exposed to Succinic acid (99.9% purity) in DMSO at concentrations of 0, 3, 10, 33, 100, 333, 666, 1000, 3333 and 10000 µg/plate by a preincubation protocol in the presence and absence of metabolic activation via Aroclor 1254 -induced male Sprague Dawley rat or Syrian hamster liver S9.

Succinic anhydride was tested up to concentrations causing cytotoxicity or exceeding the recommended limit concentration. The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background in any of the strains tested. Based on these results, Succinic anhydride is not considered to be mutagenic in bacterial cells.

The study is classified as acceptable.

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