Carboxylic acids, di-, C4-6

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Dicarboxylic acid mixture was shown to be not mutagenic in vitro in a bacterial reverse mutation assay and in a mammalian cell forward gene mutation assay in CHO cells. Clastogenic effects were reported in vitro in a chromosome aberration analysis in CHO cells in the presence of metabolic activator S9 in concentrations of 1500 µg/mL and above. Since cytotoxicity as reduced cell proliferation was already seen in cultures treated with 1000 µg/mL in the pilot experiment of this test, the positive effect could be related to cytotoxicity. No effect on DNA repair in vitro was observed in an unscheduled DNA synthesis test in primary rat hepatocytes.  

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Principles of method if other than guideline:

Bibliography: Ames et al., Mut.Research. 31: 347-364, 1975
Appropriate positive controls included

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

test item: AGS-Mixture (Adipic acid: 6-9%; Glutaric acid 30-35%; Succinic acid 8-11%; Nitric acid 0.5 - 3%) used as 50% aqueous solution

Target gene:

Histidine auxotrothy

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix obtained from Aroclor 1254-treated Sprague Dawley rats

Test concentrations with justification for top dose:

30, 100, 300, 1000, 3000 µg/plate
These dose levels were determined in a preliminary toxicity screen with 10, 167, 500, 1667 and 5000 µg/plate.

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Positive control substance:

other: Sodium acide, 9-aminoacridine, 2-nitrofluorene, 2-anthramine

Details on test system and experimental conditions:

plate incorporation assay

Evaluation criteria:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies with at least one dose point inducing a mutant frequency value that is two-fold the solvent control value.

Statistics:

Significance at the 95% conficence limits is determined by the program developed by Moore and Felton (1983).

Species / strain:

other: all strains tested

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

Remarks on result:

other: all strains/cell types tested

Bacterial toxicity was investigated in a pilot experiment; no relevant colonies at 5000 µg/plate; high dose for main experiment: 3000µg/plate. All solvent and positive controls showed the expected results.

The results of the test article, AGS Mixture, were negative in strains TA 1535, 1537, 98 and 100 of S. typhimurium with and without metabolic activation at all doses tested (highest dose: 3000 µg/plate).

In the initial assay, strain 1538 exhibited an increase in mutant frequency at the hghest dose (2.5 fold above the concommitant control) without metaboilic activation. Two subsequent retests failed to confirm the initial observation (see Table 1)

Overall the authors concluded that the test compound is not mutagenic in the Ames test.

Table 1: summary of results

dose level (µg/plate)	S9 -mix ( + or -)	TA1535	TA1537	TA98	TA100	TA1538 (1.exp)	TA1538 (2. exp)	TA1538 (3.exp)
water	-	14	13	28	141	14	12	12
water	+	9	11	44	159	20	-	-
sodium acide, 10	-	1107	-	-	1152	-	-	-
9 -Aminoacridine, 150	-	-	1496	-	-	-	-	-
2 -Nitrofluorene, 5	-	-	-	486	-	507	446	551
2 -Anthramine, 2.5	+	250	232	1544	1467	1436	-	-
AGS mix, 30	-	17	12	26	150	13	6	12
AGS mix, 100	-	13	10	24	133	15	8	12
AGS mix, 300	-	16	12	24	144	15	5	9
AGS mix, 1000	-	14	15	32	148	15	11	16
AGS mix, 3000	-	17	14	42	140	35	21	11
AGS mix, 30	+	10	10	38	119	18	-	-
AGS mix, 100	+	11	10	42	188	25	-	-
AGS mix, 300	+	8	13	34	190	13	-	-
AGS mix, 1000	+	8	18	43	181	31	-	-
AGS mix, 3000	+	11	14	42	197	30	-	-

AGS mixture was tested as 50% aqueous solution.

Executive summary:

Mutagenic activity of dicarboxylic acid mixture (AGS-Mixture, 50%) was investigated in a study similar to OECD TG 471 in bacteria (Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538) with or without metabolic activator S9. Cytotoxicity was observed at 5000 µg/plate. Negative and positive controls were functional in all experiments (Barfknecht 1986, BASF 1990).

In the initial assay, strain TA1538 exhibited a dose related increase in mutant frequency that reached 2.5-fold the solvent control background value without metabolic activation. However, two subsequent retests failed to confirm the initial observation. All other tests were negative. The authors concluded that dicarboxylic acid mixture is not mutagenic in the 5 strains tested with and without metabolic activation at dose levels of 30, 100, 300, 1000 and 3000 µg/plate.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only 4 Salmonella strains used

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

neither TA102 or WP2uvrA strains were used to detect cross-linking mutagens

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine auxotrophy

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S-9 mix

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500 and 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqua dest

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 10 µg of 2-aminoanthracene for all strains

Remarks:

with S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 5 µg of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for TA 100 and TA 1535; 10 µg of 4-nitro-o-phenylendiamine for TA 98; 100 µg of 9-aminoacridine chloride monohydrate for TA 1537

Remarks:

without S9 mix

Details on test system and experimental conditions:

- The standard plate test (SPT) was performed according to the method of Ames et al. (Proc. Nat. Acad. Sci. USA, 70, 2281 - 2285, 1973; Mut. Res., 31, 347 - 364, 1975) and the Preincubation test (PIT) according to the method of Yahagi et al. (Mut. Res., 48, 121 - 130, 1977) and Matsushima et al. (In: Norpoth, K.H. and R.C. Garner, Springer Verlag Berlin, Heidelberg, New York, 1980).
- 3 test plates per dose

Evaluation criteria:

A substance to be characterized as positive has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at doses 2500 µg/plate onwards

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Incomplete solubility of test substance in aqua dest from about 2500 µg/plate onward

Remarks on result:

other: all strains/cell types tested

Remarks:

Salmonella typhimurium; TA1535 TA100 TA1537 TA98

Standard Plate Test: Revertants/plate (mean ± SD)

Dose (µg/ml)	TA 98		TA 100		TA 1535		TA 1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	31±4	43±4	101±3	115±6	15±3	15±3	9±2	11±1
20	37±7	43±4	117±6	122±4	16±3	16±4	9±1	11±3
100	31±2	39±5	119±16	121±29	16±5	12±6	14±6	8±3
500	27±7	37±8	115±11	89±4	12±1	14±2	12±2	14±7
2500*	34±5	18±7	91±9	61±9	17±7	15±4	13±5	12±2
5000*	31±3	18±6	63±2	39±4	10±2	14±2	11±4	10±1
Positive control	890±36	1517±126	1467±115	1217±161	1290±271	223±32	330±36	143±12

 

Preincubation Test: Revertants/plate (mean ± SD)

Dose (µg/ml)	TA 98		TA 100		TA 1535		TA 1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	21±7	37±1	98±8	110±16	23±2	20±2	11±2	1±2
20	21±2	38±6	103±16	123±9	34±4	28±1	11±3	21±5
100	21±3	40±5	115±5	127±8	31±2	24±7	9±3	18±3
500	21±10	44±3	106±8	126±3	35±5	23±2	13±2	18±0
2500*	18±4	33±2	48±14	91±6	22±4	24±2	15±1	11±5
5000*	21±4	25±1	57±28	66±6	8±1	13±4	11±1	7±4
Positive control	833±32	923±223	1340±193	1310±272	1370±30	240±10	450±61	333±21

* precipitation

Executive summary:

A bacterial mutagenicity test on Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 gave no indication of a mutagenic potential for dicarboxylic acids.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

triplicate assays were conducted; concomitant positive control compounds were included

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

test item: AGS-Mixture (Adipic acid: 6-9%; Glutaric acid 30-35%; Succinic acid 8-11%; Nitric acid 0.5 - 3%) used as 50% aqueous solution

Target gene:

HPRT forward mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver homogenate (S9 mix); for the main experiment 10% S9 mix was used

Test concentrations with justification for top dose:

1500, 1750, 2000, 2250, 2500 µg/ml without S9-mix (tested as 50% aqueous solution)
1500, 2000, 2500, 3000, 3250 µg/ml with S9-mix (tested as 50% aqueous solution)

Vehicle / solvent:

AGS mixture is used as 50% aqueous solution. No adjustion for content was made in the formulation of doses. All required dilutions were made with sterile water on the day of the test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5 x 10 E5 cells in 5 mL medium per flask
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 hours
- Harvest time after the end of treatment (sampling/recovery times): 19 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7-8 days
- Selection time (if incubation with a selective agent): 7 days
- Selective agent: 6-thioguanine
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2 x 10 E5 cells

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:
- Any supplementary information relevant to cytotoxicity:

Evaluation criteria:

The mutation frequency was calculated by correcting the total number of mutant colonies by the cloning efficiency of the cells at the time of mutant selection.

Statistics:

Dose-response analyses were performed on transformed muation frequency data by the one-way analysis of variance method outlined by Snee and Irr (1981). Dose response was considered significant if (p >= 0.01). A computer program obtained from Monsanto company used to analyse the data.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations above 3000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

No statistical significant differences were observed. All positive and negative controls were within acceptable range. Cloning efficiencies in the high doses were approximately 80 % both in the presence and absence of metabolic activation

Conclusions:

in vitro gene mutation assay (HPRT Test): negative

Executive summary:

The test article, AGS mixture, was tested as 50% aqueous solution in the gene mutation assay in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay at dose levels of 1500, 1750, 2000, 2250, 2500 µg/ml without metabolic activation and at dose level of 1500, 2000, 2500, 3000 and 3250 µg/ml in the presence of metabolic activation (S9 -mix preparation). These concentrations were based on pilot experiments to determine cytotoxicity. Relative initial survival in the high doses were approximately 10 -20 % both in the presence and absence of metabolic activation. There were no stastistically significant increases in the mutation frequencies of AGS Mixture (50%) treated cultures when compared to the negative/solvent controls. Thus, the test article was negative in the CHO/HPRT Test under the conditions and according to the criteria of the test protocol.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

limited data on purity of test compound

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

test item: AGS-Mixture (Adipic acid: 6-9%; Glutaric acid 30-35%; Succinic acid 8-11%; Nitric acid 0.5 - 3%) used as 50% aqueous solution

Target gene:

Chromosome abberations

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4

Cytokinesis block (if used):

colcemid (2 x 10 E-7 M final concentration) added 2-3 hours prior to preparation of cells for metaphase analysis

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix from Arochlor 1254 induced rat liver

Test concentrations with justification for top dose:

In a preliminary test dose levels of 100, 250, 500, 750, 1000, 2000, 4000, 5000, and 11500 µg/mL with and without S9-mix were evaluated. Based on the cell proliferation kinetics and the toxic effect of the test article, the dose levels selected for analysis were:
100, 750, and 1000 µg/ml without S9-mix and 200, 800, and 2000 µg/mL with S9-mix

Vehicle / solvent:

distilled water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitro-N-nitrosoguanidine (1.5 µg/mL) and N-nitrosodimethylamine (1000 µg/mL)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (duplicate)
- Number of independent experiments : one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.8 x 10 €6 cells in medium (15 mL)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 16-22 hours
- Exposure duration/duration of treatment: 5 hours
- Harvest time after the end of treatment (sampling/recovery times): 14-18 hours (including 2 hours cell cycle arrest)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colcemid in a concentration of 2 x 10 E-7 M
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Giemsa
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 100
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): based on chromosome number (+/- 2 of the modal no. of 20) and chromosome morphology; chroomosome aberrations are classified according to Buckton and Evans, 1973 and Savage, 1975

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cell proliferation kinetics as a measure of mitotic delay; average proliferation time (evaluation for first, second and third division metaphases and average proliferation time

Evaluation criteria:

Assessment of a test arbicle as positive is based upon its ability to produce a statistically significant iincrease in chromosome aberrations in test cultures, as compared to to the solven tcontrols in at least one of the test points, and/or its ability to produce a dose response.

Statistics:

The results are subjected to statistical analysis. The T-test is used to compare pairwise the treatment group with the control group using the number of aberrations per cell. The proportion of aberrant metaphases will be analysed using the Chi-square analysis.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at concentrations ≥ 1500 µg/mL with S9-mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

In a preliminary test dose levels of 100, 250, 500, 750, 1000, 2000, 4000, 5000, and 11500 µg/mL with and without S9-mix were evaluated. Based on reduced cell proliferation kinetics starting at 750 µg/mL (-S9) or 1000 µg/mL (+S9) and overall toxicity at ≥ 2000 µg/mL (-S9) or ≥ 4000 µg/mL (+S9) the following dose levels were tested in the main experiment:

without S9 -mix: 100, 750 and 1000 µg/ml

with S9 -mix: 200, 800 and 2000 µg/ml

A statistical significant increase in the number of aberrations per cell and the proportion of aberrant metaphases was observed at 2000 µg/ml in the presence of S9 -mix. No increases were observed at any dose level without metabolic activation.

To elucidate the biological significance of this positive finding, the test article was re-evaluated with S9 -mix: 

with S9-mix: 1500, 2000, and 2200 µg/mL

Statistically significant increases in aberrations per cell and proportion of aberrant metaphases were noted at all dose levels re-evaluated. Thus the test item was considered positive in the in vitro chromosomal aberration assay at concentrations ≥ 1500 µg/ml. Since cytotoxicity as reduced cell proliferation was already seen in cultures treated with 1000 µg/mL in the pilot experiment, the positive effect could be related to cytotoxicity. (see attachment)

Executive summary:

Dicarboxylic acid mixture was investigated in an in vitro Chromosome Aberration Analysis in CHO cells. Dose levels of 100, 250, 500, 750, 1000, 2000, 4000, 5000 and 11500 µg/ml were evaluated with and without metabolic activation in a pilot experiment. Based on cytotoxicity data the dose level selected for analysis in the main experiment were 100, 750 and 1000 without S9 and 200, 800 and 2000 µg/ml with S9.

A statistically significant increase was observed in the number of aberrations per cell and in the proportion of aberrant metaphases at the 2000 µg/ml dose level with metabolic activation. No increases were observed at any dose level without metabolic activation. To elucidate the biological significance of this positive finding, the test article was re-evaluated with S9 at doses of 1500, 2000, 2200 µg/ml. Statistically significant increase in aberrations per cell and proportion of aberrant metaphases were noted at all dose levels re-evaluated. Thus the test item was considered positive in the in vitro chromosomal aberration assay at concentrations ≥ 1500 µg/ml. Since cytotoxicity as reduced cell proliferation was already seen in cultures treated with 1000 µg/mL in the pilot experiment, the positive effect could be related to cytotoxicity.

Genetic toxicity in vivo

Description of key information

The potential of dicarboxylic acid mixture to induce structural chromosomal aberrations in vivo was tested in hematopoetic cells of the rat bone marrow after oral gavage. No in vivo clastogenic effect was observed. 

Overall, dicarboxylic acid mixture does not exert gene mutations in vitro in bacteria and mamalian cells. The clastogenic effects seen at high and cytotoxic concentrations in vitro could not be reproduced in vivo. This overall negative conclusion is confirmed by mutagenicity studies on the constituents of dicarboxylic acid mixture with negative outcome.

In conclusion, there is no relevant mutagenic potential of dicarboxylic acid mixture.

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:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

AGS mixtures were administered in a single oral dose to SD rats. Animals were sacrificed 6, 18 or 30 h after treatment. A positive control group was included (Cyclophosphamide). 2 hours before scheduled sacrifice animals were administered colchicine at to arrest cells in metaphase. Animals were sacrificed and both femurs were removed from each animal and metaphase slides were prepared. Slides were stained, coded and scored for chromosomal aberrations (50 metaphase bone marrow cells/animal were evaluated.

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Specific details on test material used for the study:

test material: AGS mixture was used as a 50% aqueous solution.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

breeder: Charles River Lab., Wilmington, Massachsusetts

Sex:

male/female

Details on test animals or test system and environmental conditions:

body weight at randomization. 239-264 g for males and 178-208 g for females

Route of administration:

oral: gavage

Vehicle:

deionized water

Details on exposure:

all animals were fasted overnight prior to treatment; AGS mixture (50% aqueous solution), solvent or positive controls were administered as single oral doses at 5 mL/kg bw.
2 to 3 hours prior to sacrifice each animal was given a single intraperitoneal dose of colchicine at 4 mg/kg bw to arrest dividing cells in metaphase. Colchicine was dosed at 10 mL/kg bw and was prepared in distilled water.

Justification for doses:
AGS mixture (50%) was evaluated in a preliminary study at doses of 1375, 2750 and 5500 mg/kg bw. Due to the mortality and pharmacotoxic signs observed at 5500 mg/kg bw for males and 2750 mg/kg bw for females, doses selected for evaluation in the Metaphase Analysis Assay were 2750 mg/kg bw for males and 1375 mg/kg bw for females as an estimation of the maximum tolerated dose.
5500 mg/kg bw: both treated females died within four hours post dosing; one male died 24 hours post dosing and the other exhibited severe pharmacotoxic signs through day 7
2750 mg/kg bw: one female died 24 hours post dosing and the second female died by day 3; both males survived the treatment and exhibited mild to severe pharmacotoxic signs through day 7
Additional tests with higher dilutions (same doses) showed similar effects.

Duration of treatment / exposure:

6, 18, 30 h

Frequency of treatment:

single treatment

Dose / conc.:

1 375 mg/kg bw (total dose)

Remarks:

dose for females, sacrifice 6, 18, and 30 hours after treatment

Dose / conc.:

2 750 mg/kg bw (total dose)

Remarks:

dose for males, sacrifice 6, 18, and 30 hours after treatment

No. of animals per sex per dose:

vehicle control (water): 7 males and 5 females (6 hour treatment), 5 males and 5 females (each 18 hour and 30 hour treatment)
AGS mixture (50%), 2750 mg/kg bw: 8 males (6 hour treatment), 5 males (each 18 hour and 30 hour treatment)
AGS mixture (50%): 1375 mg/kg bw: 5 females (each 6, 18 hour and 30 hour treatment)
Cyclophosphamide: 20 mg/kg bw: 5 males and 5 females (18 hour treatment)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

Animals were sacrificed and both femurs were removed from each animal and metaphase slides were prepared.
A total of 500 well spread metaphase cells with a minimum of
overlapping chromosomes, were scored for the presence of chromosome aberration
per experimental treatment point (50 per animal) by two investigators (25 each
per animal). A total of 600 metaphases (12 animals) were scored the 6 hour
solvent control group. Cells were located by systematic searching of the
slide under low power (20X-40X) magnification. Cells judged acceptable for
analysis based on cell morphology and total chromosome nurnber (± 2 of the
normal diploid no. of 42) were then further analyzed with lOOX oil immersion
objective where abnormalities were detected and classified. Vernier
coordinates were recorded for the first and last metaphase scored, as well as
for any abnormal rnetaphases (including gaps) observed. The centromere number
was recorded on all cells analyzed.

Evaluation criteria:

Cytogenetic abnormalities were classified on a standard
scoring sheet according to chromosome or chromatid aberrations and further
according to type of aberration (see Legend on page 12). Aberrations were
classified according to the nomenclature cf Buckton and Evans, 1973 and
Savage, 1975.

Statistics:

Mean aberrations per cell per rat (50 cells per rat)
were analyzed for statistically significant increases in chromosome aberration
by one-tailed t tests. Each treatment group was analyzed separately as
compared to its concurrent negative control group. The mean and standard
deviation of aberrations/cell were also determined for each group of rats (500
cells~ 50 cells per rat). The number of aberrant metaphases was analyzed by
Chi-square analysis for statistically significant increases. Statistical
significance was determined at the p >= 0.05 probability level.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

death in high dose group

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Doses selected for evaluation in the Metaphase Analysis Assay were 2750 mg/kg bw for males and 1375 mg/kg bw for females as an estimation of the maximum tolerated dose.

General toxicity in the main assay:

Three males died before sacrifice; most of the remaining males and females had decreased body tone and activity, body drop, abnormal gait, ptosis and piloerection. In addition, some animals had tremors and vocalization on touch.

6 Hour Group - (observed approximately 4 hours after dosing):
One male was dead before colchicine administration. The remaining males had decreased body tone and activity, body drop, abnormal gait, ptosis and piloerection. In addition, two males bad tremors. All females showed bad decreased body tone, piloerection and vocalization on touch. A second male died before harvest. The sponsor was notified and requested that 5 extra male rats be dosed, two with the solvent control and the other three with AGS mixture (50%) at 2750 mg/kg to ensure two available rats to replace the dead ones. One male died before colchicine administration. The other two males exhibited moderate pharmacotoxic signs.

18 Hour Group - (observed approximately 16 hours after dosing):
All rats had piloerection. Three males and three females also had decreased body tone. Four males and three fernales made vocalizations when touched. One male bad abnormal gait and one female abnormal stance.

30 Hour Group - (observed approximately 28 hours after dosing):
All rats had decreased body tone and piloerection. All males had abnormal stance. One male and two females made vocalizations when touched. Two fernales had body drop.

No statistically significant increase in the incidence of aberrations or the number of cells with one or more aberrations were observed in the animals treated with dicarboxylic acid mixture at any dose and at any of the three sampling time points. (see Table 1 and attachment)

 

Table 1: in vivo bone marrow cytogenetics - proportion of cells with one ore more aberrations

 

compound	dose (mg/kg bw)	harvest time (hrs)	sex	no. of rats	no. of cells with one or more aberrations	no. of normal cells	% aberrant cells/group
water	5 mL/kg bw	6	M	7	1	349	0.3
water	5 mL/kg bw	6	F	5	1	249	0.4
AGS mix (50%)	2750	6	M	5	1	249	0.4
AGS mix (50%)	1375	6	F	5	0	250	0.0
water	5 mL/kg bw	18	M	5	2	248	0.8
water	5 mL/kg bw	18	F	5	4	246	1.6
AGS mix (50%)	2750	18	M	5	1	249	0.4
AGS mix (50%)	1375	18	F	5	0	250	0.0
cyclophosphamide	20	18	M	5	123	127	49.2**
cyclophosphamide	20	18	F	5	128	122	51.2**
water	5 mL/kg bw	30	M	5	0	250	0.0
water	5 mL/kg bw	30	F	5	1	249	0.4
AGS mix (50%)	2750	30	M	5	2	248	0.8
AGS mix (50%)	1375	30	F	5	1	249	0.4

** significant in Chi-square at p >= 0.01

Executive summary:

The potential of dicarboxylic acid mixture (50%) to induce structural chromosomal aberrations in the hemopoetic cells of the rat bone marrow were investigated in vivo after oral gavage. Dicarboxylic acid mixture (2750 mg/kg bw for males and 1375 mg/kg bw for females) was administered in single oral doses to SD rats and animals were sacrificed 6, 18 and 30 hours after dosing. A positive control group (cyclophosophamide; 20 mg/kg) was sacrificed at the 18 hours time point. 2 Hours before sacrifice animals were administered with colchinine to arrest cells in metaphase. Both femurs were removed from each animal and metaphase slides were prepared. A total of 50 metaphase  cells were analysed for each animal for the presence of chromatid and chromosome type aberrations. The positive control group resulted in a significant increase in chromosomal aberrations.

Two rats dosed with 2750 mg/kg died. The surviving animals in all test groups exhibited from mild to severe pharmacotoxic signs and the authors concluded that the test item was evaluated at or near the MTD. No statistically significant increase in the incidence of aberrations or the number of cells with one or more aberrations were observed in the animals treated with dicarboxylic acid mixture at any dose and at any of the three sampling time points. Therefore, in vivo the test item was not clastogenic to hempoietic cells of the rat bone marrow.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Mutagenic activity of dicarboxylic acid mixture (AGS-Mixture, 50%) was investigated in a study similar to OECD TG 471 in bacteria (Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538) with or without metabolic activation via rat S9. Cytotoxicity was observed at 5000 µg/plate. Negative and positive controls were functional in all experiments (Monsanto, 1986a). In the initial assay, strain TA1538 exhibited a dose related increase in mutant frequency that reached 2.5-fold the solvent control background value without metabolic activation. However, two subsequent retests failed to confirm the initial observation. All other tests were negative. The authors concluded that dicarboxylic acid mixture is not mutagenic in the 5 strains tested with and without metabolic activation at dose levels of 30, 100, 300, 1000 and 3000 µg/plate.

 

A bacterial mutagenicity test on Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 gave also no indication of a mutagenic potential for dicarboxylic acids (BASF, 1990).

 

The test article AGS-Mixture (50%) was evaluated in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay at dose levels of 1500, 1750, 2000, 2250, 2500 µg/ml without metabolic activation and at dose level of 1500, 2000, 2500, 3000 and 3250 µg/ml in the presence of metabolic activator S9. These concentrations were based on pilot experiments to determine cytotoxicity. Relative initial survival in the high doses were approximately 10 -20 % both in the presence and absence of metabolic activation. There were no statistically significant increases in the mutation frequencies of AGS Mixture treated cultures when compared to the negative/solvent controls. Thus, the test article was negative in the CHO/HPRT Test under the conditions and according to the criteria of the test protocol.

 

Dicarboxylic acid mixture was investigated in an in vitro unscheduled DNA synthesis (UDS) test to investigate a potential influence on DNA repair. The test item, AGS-Mixture (50%) was tested in concentrations ranging from 1 to 5000 µg/ml. Primary rat hepatozyte cell cultures were exposed simultaneously to the test material and ³H-thymidine. After incubation cells were washed, fixed, stained with methyl-green Pyronin and quantitative audiographic grain-conting was performed. At least 150 morphologically unaltered cells were scored for each concentration. Cytotoxicity was observed at 5000 µg/ml in the first experiment. AGS Mixture (50%) turned the medium acidic at concentrations of 1000 and 2500 µg/ml. No attempt was made to determine the precise pH at the time of exposure. UDS was measured at concentrations of the test compound between 1 and 1000 µg/ml in the first experiment and 10 and 2500 µg/ml in the second experiment. No increase in UDS was observed at any concentration measured. The sensitivity of the test system was demonstrated by positive response to 2-actylaminoflourene.

 

Dicarboxylic acid mixture was investigated in an in vitro Chromosome Aberration Analysis in CHO cells. Dose levels of 100, 250, 500, 750, 1000, 2000, 4000, 5000 and 11500 µg/ml were evaluated with and without metabolic activation. Based on cytotoxicity data the dose level selected for analysis were 100, 750 and 1000 without S9 and 200, 800 and 2000 µg/ml with S9. A statistically significant increase was observed in the number of aberrations per cell and in the proportion of aberrant metaphases at the 2000 µg/ml dose level with metabolic activation. No increases were observed at any dose level without metabolic activation. To elucidate the biological significance of this positive finding, the test article was re-evaluated with S9 at doses of 1500, 2000, 2200 µg/ml. Statistically significant increase in aberrations per cell and proportion of aberrant metaphases were noted at all dose levels re-evaluated. Thus the test item was considered positive in the in vitro chromosomal aberration assay at concentrations ≥ 1500 µg/ml. Since cytotoxicity as reduced cell proliferation was already seen in cultures treated with 1000 µg/mL in the pilot experiment, the positive effect could be related to cytotoxicity.

 

The potential of dicarboxylic acid mixture to induce structural chromosomal aberrations in the hematopoetic cells of the rat bone marrow was investigated in vivo after oral gavage. Dicarboxylic acid mixture was administered in single oral doses to SD rats. In a preliminary study with doses of 1375, 2750 and 5500 mg/kg bw mortality and pharmacotoxic signs were observed at 5500 mg/kg bw for males and 2750 mg/kg bw for females. The doses selected for evaluation in the Chromosomal Aberration Assay were thus chosen with 2750 mg/kg bw for males and 1375 mg/kg bw for females as an estimation of the maximum tolerated dose. Treated animals were sacrificed 6, 18 and 30 hours after dosing. The positive control group (cyclophosophamide; 20 mg/kg) was sacrificed at the 18 hours time point. 2 Hours before sacrifice animals were administered with colchinine to arrest cells in metaphase. Both femurs were removed from each animal and metaphase slides were prepared. A total of 50 metaphase cells were analysed for each animal for the presence of chromatid and chromosome type aberrations. The positive control group resulted in a significant increase in chromosomal aberrations.

Two rats dosed with 2750 mg/kg died. The surviving animals in all test groups exhibited from mild to severe pharmacotoxic signs and the authors concluded that the test item was evaluated at or near the MTD. No statistically significant increase in the incidence of aberrations or the number of cells with one or more aberrations were observed in the animals treated with dicarboxylic acid mixture at any dose and at any of the three sampling time points. Therefore, in vivo the test item was not clastogenic to hematpoietic cells of the rat bone marrow.

This overall negative assessment is confirmed by studies on the constituents of dicarboxylic acid mixture. Adipic acid did not exert a mutagenic potential in vitro and in vivo and also mutagenicity tests with glutaric and succinic acid were throughout negative.

Conclusion:
Dicarboxylic acid mixture was shown to be not mutagenic in vitro in a bacterial reverse mutation assay and in a mammalian cell forward gene mutation assay in CHO cells. Clastogenic effects were reported in vitro in a chromosome aberration analysis in CHO cells in the presence of metabolic activator S9 in concentrations > 1500 mg/ml. The potential of dicarboxylic acid mixture to induce structural chromosomal aberrations in vivo was tested in hemopoetic cells of the rat bone marrow after oral gavage. No in vivo clastogenic effect was observed. In addition no effect on DNA repair in vitro was observed in an unscheduled DNA synthesis test in primary rat hepatocytes.

This overall negative assessment is confirmed by mutagenicity studies on the constituents of dicarboxylic acid mixture with negative outcome.

Overall it can be concluded that dicarboxylic acid mixture exerts no relevant mutagenicity.

Justification for classification or non-classification

No classification is required according to the classificaton criteria of Regulation no. 1272/2008 (GHS).