Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In two separate in vitro laboratory studies, succinic anhydride was non-mutagenic in the Ames assay with and without metabolic activation at doses of up to 10000 ug/plate. No evidence of in vitro mutagenicity was seen when succinic anhydride was tested for chromosomal aberrations and sister chromatid exchanges in two additional laboratory studies conducted in vitro (Melnick, 1990).

No studies on in vitro gene mutation in mammalian cells with succinic anhydride could be identified from literature. Therefore, this endpoint was assessed in a read-across approach with maleic acid. Maleic acid was considered to be non-mutagenic in the HPRT assay (Wollny, 2010). Read-across is feasible because a common feature of cyclic anhydrides is that they readily hydrolyse to their corresponding dicarboxylic acids in aqueous solutions. Hydrolysis fo succinic anhydride yields succinic acid, which is a close structural analogue of maleic acid. Compared to succinic acid, maleic acid only differs in an unsaturated 2,3-carbon-carbon bond. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Link to relevant study records

Referenceopen allclose all

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
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
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%
Target gene:
histidine-dependence
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
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
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.

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             
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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
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 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 1983
Principles of method if other than guideline:
A detailed presentation of the technique for detecting chromosomal aberrations is found in Galloway et al. (1985). Briefly, Chinese hamster ovary cells were incubated with study compound or solvent as indicated. Cells were arrested in first metaphase by addition of colcemid and harvested by mitotic shake-off, fixed, and stained in 6% Giemsa.
In the absence of S9, cells were incubated with study compound or solvent for 8-10 hours at 37° C. Cells were then washed, and fresh medium containing colcemid was added for an additional 2-3 hours followed by harvest.
In the presence of S9, cells were incubated with study compound or solvent for 2 hours at 37° C. Cells were then washed, medium was added, and incubation was continued for 8-10 hours. Colcemid was added for the last 2-3 hours of incubation before harvest. S9 was from the liver of Aroclor 1254-induced male Sprague Dawley rats.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague Dawley rat liver S9 and cofactor mix
Test concentrations with justification for top dose:
500, 750, and 1000 ug/mL succinic anhydride
The high dose was limited by toxicity or solubility but did not exceed 1 mg/ml for chromosomal aberration test.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
-Without S9: In the absence of S9, cells were incubated with study compound or solvent for 8 hours at 37° C. Cells were then washed, and fresh medium containing colcemid was added for an additional 2 hours followed by harvest.
-With S9: In the presence of S9, cells were treated with the study chemical and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with colcemid present for the final 2 hours. of incubation before harvest. S9 was from the liver of Aroclor 1254-induced male Sprague Dawley rats.
-Cells were harvested by mitotic shake-off, fixed and stained in 6% Giemsa. The harvest time for the chromosomal aberration test was based on the cell cycle information obtained in the SCE test; if cell cycle delay was anticipated, the incubation period was extended approximately 5 hours.
Evaluation criteria:
Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 +/- 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. 100 first-division metaphase cells were scored at each dose for the chromosomal aberration test. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).
Statistics:
Statistical analyses were conducted on both the slopes of the dose-response curves and the individual dose points. Chromosomal aberration data are presented as percentage of cells with aberrations. As with SCEs, both the dose-response curve and individual dose points were statistically analyzed. A statistically significant (p<0.003) trend test or a significantly increased dose point (p<0.05) was sufficient to indicate a chemical effect.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1: Induction of chromosomal aberrations in CHO cells by succinic anhydride

             -S9Harvest time: 10.5 hours              +S9Harvest time: 12.5 hours
 Dose (ug/mL)  Total cells  No. of Abs  Abs/cell  % cell with Abs  Dose (ug/mL)  Total cells  No. of Abs  Abs/cell  % cells with Abs

DMSO 

100
100  0.01  1.0 

DMSO

100 
100  0.02  2.0 

Succinic anhydride 

500

100  0.01 

1.0 

Succinic anhydride

500 

100  0.04   4.0
 750 100  0.03  3.0   750  100  0.04 3.0 
 1000 100  0.02   1.0  1000  100  4 0.04  3.0 
 Summary Negative           Summary  Negative          

Mitomycin C

0.5 
100  0.06  5.0 

Cyclophos-

phamide

7.5 
100   10  0.10 9.0 
 1.0 25  0.32  28.0  37.5  25  15 0.60  40.0 
Conclusions:
Under the experimental conditions reported, succinic anhydride is considered to be clastogenic in an in vitro Chromosome Aberration Test with Chinese Hamster Ovary Cells.
Executive summary:

In a mammalian cell chromosomal aberration test conducted similar to OECD TG 473, CHO cell cultures were exposed to succinic anhydride in DMSO at concentrations of 500, 750 and 1000 µg/ml with and without metabolic activation with S9 from the liver of Aroclor 1254-induced male Sprague Dawley rats.

Positive controls induced the appropriate response in the assay. There was no evidence of chromosome aberrations or sister chromatid exchanges induced over background.

The study is classified as accepatable.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Version / remarks:
adopted 23 October 1986
Principles of method if other than guideline:
A detailed description of the SCE protocol is presented by Galloway et al. (1985). Briefly, Chinese hamster ovary cells were incubated with study compound or solvent as described and cultured for sufficient time to reach second metaphase division. Cells were then collected by mitotic shake-off, fixed, air dried, and stained.
In the absence of S9, Chinese hamster ovary cells were incubated with study compound or solvent for 2 hours at 37° C. Then BrdU was added, and incubation was continued for 24 hours. Cells were washed, fresh medium containing BrdU and colcemid was added, and incubation was continued for 2-3 hours.
In the presence of S9, cells were incubated with study compound or solvent for 2 hours at 37° C. Cells were then washed, and medium containing BrdU was added. Cells were incubated for a further 26 hours, with colcemid present for the final 2-3 hours. S9 was from the liver of Aroclor 1254-induced male Sprague Dawley rats.
GLP compliance:
yes (incl. QA statement)
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague Dawley rat liver S9 and cofactor mix
Test concentrations with justification for top dose:
50, 166.5, and 500 ug/mL succinic anhydride. The high dose was limited by toxicity or solubility but did not exceed 0.5 mg/ml for SCE test
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
-Without S9: In the absence of S9,cells were incubated for 26 hours with the study chemical in McCoy's 5A medium supplemented with 10% fetal bovine serum, L-glutamine (2 mM), and antibiotics. BrdU was added 2 hours after culture initiation. After 26 hours, the medium containing the study chemical was removed and replaced with fresh medium plus BrdU and colcemid, and incubation was continued for 2 more hours.
-With S9: In the presence of S9,cells were incubated with the chemical, serum free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing BrdU and no study chemical; incubation proceeded for an additional 26 hours, with colcemid present for the final 2 hours. S9 was from the liver of Aroclor 1254-induced male Sprague Dawley rats.
-Cells were harvested by mitotic shake-off, fixed and stained with Hoechst 33258 and Giemsa.
If significant chemical-induced cell cycle delay was seen, incubation time was lengthened to ensure a sufficient number of scorable cells.


Evaluation criteria:
Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 +/- 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. For the SCE test, 50 second-division metaphase cells were usually scored for frequency of SCEs per cell from each dose.
Statistics:
Statistical analyses were conducted on both the slopes of the dose-response curves and the individual dose points. An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less than 0.001. A statistically significant (p<0.003) trend test or a significantly increased dose point (p<0.05) was sufficient to indicate a chemical effect.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1. Induction of sister chromatid exchanges in CHO cells by succinic anhydride

 Compound  Dose (ug/mL)  Total cells  No. of chromosomes  No. of SCEs  SCEs/ chromosome  SCEs/ cell  Hours in BrdU  Relative SCEs/Cell (%)a

                         -S9

Summary: Negative

 DMSO

   50 1033  411  0.4  8.2  25.5   
 Succinic anhydride  50 50   1033 362  0.35  7.2  25.5  87.8 
   166.5 50  1039  392  0.38  7.8  25.5  95.1 
   500 50  1040  369  0.35  7.4  25.5  90.2 
 Mitomycin C 0.001  50  1017  509  0.50  10.2  25.5  124.4 
  0.01  105  200  1.90  40.0  25.5  487.8 

                         +S9

Summary: Negative

 DMSO   50  1045  384  0.37  7.7  25.5   
 Succinic anhydride  50  50  1041  424  0.41  8.5 25.5  110.4 
  166.5  50  1040  401  0.39  8.0  25.5  103.9 
  500  50  1039  372  0.36  7.4  25.5  96.1 
 Cyclophos- phamide  0.4 50  1035  711  0.69  14.2  25.5  184.4 
   2 104  181  1.74  36.2  25.5  470.1 

a SCEs/cell of culture exposed to study chemical relative to those of culture exposed to solvent

Conclusions:
Under the experimental conditions reported, succinic anhydride is considered to be clastogenic in an in vitro Sister Chromatid Exchanges Test with chinese hamster ovary cells.
Executive summary:

In a mammalian cell sister chromatide exchange test conducted similar to OECD 479, CHO cell cultures were exposed to Succinic anhydride in DMSO at concentrations of 0, 50, 166.5 and 500 µg/ml with and without metabolic activation with S9 from the liver of Aroclor 1254-induced male Sprague Dawley rats.

Positive controls induced the appropriate response in the assay. There was no evidence of sister chromatid exchanges induced over background.

The study is classified as accepatable.

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:
For details and justification of read-across please refer to the report attached in section 13 of IUCLID.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 900 µg/mL and above in experiment II without metabolic activation following 24 hours of exposure
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no
RANGE-FINDING/SCREENING STUDIES: yes, see above
COMPARISON WITH HISTORICAL CONTROL DATA: yes, see also attachment

For Tables on Results, please refer to section "Attached background material".

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix: 37.5; 75.0; 150; 300; 600; and 1200 µg/mL

with S9 mix: 37.5; 75.0; 150; 300; 600; and 1200 µg/mL

Experiment II:

without S9 mix: 75.0; 150; 300; 600; 900; and 1200 µg/mL

with S9 mix: 75.0; 150; 300; 600; 900; and 1200 µg/mL

The maximum concentration of the test item in the main experiments is equal to molar concentration of approximately 10 mM.

No precipitation of the test item was observed up to the maximal concentration in all ex-periments.

Cytotoxic effects as indicated by a relative cloning efficiency 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/10E6 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 both experiments of this study (with and without S9 mix) the range of the solvent controls was from 10.7 up to 28.5 mutants per 10E6 cells; the range of the groups treated with the test item was from 9.2 up to 59.1 mutants per 10E6 cells.

EMS (225 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:
Under the experimental conditions reported the test item maleic acid did not increase the frequency of gene mutations at the HPRT locus in V79 cells. Therefore, maleic acid is considered to be non-mutagenic in this HPRT assay.
Executive summary:

In a mammalian cell gene mutation assay using the HPRT locus, V79 cells of the chinese hamster were exposed to Maleic acid (99.7% purity) in deionised water. The assay was performed in two independent experiments, using two parallel cultures each at concentrations of 37.5, 75.0, 150, 300, 600 and 1200 µg/mL (experiment I) and 75.0, 150, 300, 600, 900 and 1200 µg/mL (experiment II).

The first main experiment was performed in the presence and absence of Phenobarbital/ß-naphthoflavone induced rat liver S-9 mix and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

Maleic anhydride was tested up to cytotoxic concentrations of 1200 µg/ml, corresponding to a molar concentration of 10 mM.

The positive controls ethylmethane sulfonate at final concentrations of 1.8 (exp. I) and 1.2 mM (exp. II) (without metabolic activation) and 7,12 -dimethylbenz(a)anthracene at a final concentration of 4.3 µM (with metabolic activation) did induce the appropriate response.

There was no evidence of induced mutant colonies or a concentration related positive response over background after treatment with the test material.

This study is classified as acceptable. This study satisfies the requirement for OECD TG 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

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 information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

According to Annex VIII, Column 2, point 8.4 of Regulation 1907/2006, provision of information on genetic toxicity in vivo is not required since all tests on in vitro endpoints were negative (Gene mutation in bacterial cells, cytogenicity in mammalian cells and gene mutation in mammalian cells).

Information from a study in Japanese on genetic toxicity indicated negative results (no genotoxicity) from in vivo screening tests of for the bone marrow test in rats (in vivo, chromosome aberrations) and silk worms (mutations) (Kawachi et al.,1980). However, the study was disregarded due to limited documentation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

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.

 

In a mammalian cell chromosomal aberration test conducted similar to OECD TG 473, CHO cell cultures were exposed to Succinic anhydride in DMSO at concentrations of 500, 750 and 1000 µg/ml with and without metabolic activation with S9 from the liver of Aroclor 1254-induced male Sprague Dawley rats.

In addition, in a mammalian cell sister chromatid exchange test conducted similar to OECD 479, CHO cell cultures were exposed to Succinic anhydride in DMSO at concentrations of 0, 50, 166.5 and 500 µg/ml with and without metabolic activation with S9 from the liver of Aroclor 1254-induced male Sprague Dawley rats.

Positive controls induced the appropriate response in both assays.

There was no evidence of chromosome aberrations or sister chromatid exchanges induced over background.

 

In addition, in a mammalian cell gene mutation assay on the HPRT locus used in a read-across approach, V79 cells of the Chinese hamster were exposed to Maleic acid (99.7% purity) in deionised water. The assay was performed in two independent experiments, using two parallel cultures each at concentrations of 37.5, 75.0, 150, 300, 600 and 1200 µg/mL (experiment I) and 75.0, 150, 300, 600, 900 and 1200 µg/mL (experiment II).

The first main experiment was performed in the presence and absence of Phenobarbital/ß-naphthoflavone induced rat liver S-9 mix and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

Maleic anhydride was tested up to cytotoxic concentrations of 1200 µg/ml, corresponding to a molar concentration of 10 mM.

The positive controls ethylmethane sulfonate at final concentrations of 1.8 (exp. I) and 1.2 mM (exp. II) (without metabolic activation) and 7,12 -dimethylbenz(a)anthracene at a final concentration of 4.3 µM (with metabolic activation) did induce the appropriate response.

There was no evidence of induced mutant colonies or a concentration related positive response over background after treatment with the test material.

This study is classified as acceptable. This study satisfies the requirement for OECD TG 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

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.

 

According to Annex VIII, Column 2, point 8.4 of Regulation 1907/2006, provision of information on genetic toxicity in vivo is not required since all tests on in vitro endpoints were negative (Gene mutation in bacterial cells, cytogenicity in mammalian cells and gene mutation in mammalian cells).

Information from a study in Japanese on genetic toxicity indicated negative results (no genotoxicity) from in vivo screening tests of for the bone marrow test in rats (in vivo, chromosome aberrations) and silkworms (mutations) (Kawachi et al.,1980). However, the study was disregarded due to limited documentation.

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 succinic anhydride is considered negative for mutagenicity. Therefore do not trigger classification according to the CLP criteria as set ou in Regulation (EC) 1272/2008.