Tetrahydromethylphthalic anhydride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance tetrahydromethylphthalic anhydride (MTHPA) was investigated for genotoxic effects for 3 different endpoints. It was shown to be not mutagenic in the Ames test with and without metabolic activation (S9). A first chromosome aberration test with CHL/IU cells revealed equivocal results. Thus a second state of the art chromosome aberration test with V79 cells was conducted. In this test MTHPA was shown to be not clastogenic. Additionally it could be shown in a mouse lymphoma assay, that MTHPA has not the potential to induce mutations in mammalian cells.

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:

1995-12-08 to 1997-07-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Guidelines for screening Mutagenicity testing of Chemicals (Japan) and OECD Test Guideline 471 and 472

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

not indicated

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: provided by Dr. A. N. Ames, California University, USA on 1975-10-31
- Properly maintained: yes, -80 °C
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: provided by Dr.Gada, National Institute of Genetics, Japan on 1979-5-9
- Properly maintained: yes, -80 °C
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

-S9 mix; 0, 62.5, 125, 250, 500, 1000, 2000 µg/plate
+S9 mix; 0, 156, 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: well-known solvent for general purpose

Details on test system and experimental conditions:

Type: Ames test
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:2

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The test substance is considered to be positive for mutagenic activity when assay plates with the test substance show a significant increase in revertant colony count as compared with that on negative control plates and when this effect is reasonably reproducible or dose dependent.

Evaluation criteria:

no details given

Statistics:

no details given

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

This chemical did not induce mutations in the S. typhimurium and E. coli strains.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.4

no remarks

Conclusions:

Tetrahydromethylphthalic anhydride (MTHPA) was not mutagenic in Salmonella typhimuriumTA100, TA1535, TA98, TA1537 and Escherichia coli WP2uvrA at concentrations up to 5 mg/plate or 2 mg/plate, with or without an exogenous metabolic activation system, respectively.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA100, TA1535, TA98, TA1537 of S. typhimurium and Escherichia coli Wp2 uvrA were exposed to tetrahydromethylphthalic anhydride in DMSO at concentrations of 0, 62.5, 125, 250, 500, 1000, 2000 ug/plate (-S9 mix) and 0, 156, 313, 625, 1250, 2500, 5000 ug/plate (+S9 mix) in the presence and absence of mammalian metabolic activation (S9 mix, Rat liver, induced with Phenobarbital and 5,6-benzoflavone).

 

Tetrahydromethylphthalic anhydride was tested up to limit concentration (5000 µg/plate). No cytotoxicity occurred. The test chemical did not induce mutations in the S. typhimurium and E. coli strains The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable. This study satisfies the requirement for the Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals Test Guideline and OECD 471 and 472 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2009-09-14 to 2009-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

The mouse lymphoma assay (MLA), employing the tk+/- (thymidine kinase) locus in L5178Y cells has been used.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection.
Cells are stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma.
For each experiment, one vial was thawed rapidly, the cells were diluted in RPMI 10 medium and incubated at 37 +/- 1 °C in a humidified atmosphere containing approximately 5 % CO2 in air. Subcultures were established in an appropriate number of flasks from well-growing cells.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The test item was investigated in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Test concentrations with justification for top dose:

Assay 1:
3-hour treatment, in absence of exogeneous metabolic activation (-S9):
5.12; 12.8; 32; 80; 140 and 200 μg/mL.
3-hour treatment, in presence of exogeneous metabolic activation (+S9):
38.4; 96; 240; 600; 1200 and 1800 μg/mL.
Assay 1 (additional test):
3-hour treatment (-S9 Mix): 80; 140; 200; 400; 800 and 1200 μg/mL.
Assay 2:
3-hour treatment (+S9 Mix): 96; 240; 600; 1200; 2000 and 3000 μg/mL.
24-hour treatment (-S9 Mix): 25.6; 64; 160; 280; 400 and 800 μg/mL.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

Assay 1.:
A 3-hour treatment was performed in the presence and absence of S9 Mix.

Assay 2.:
A second assay was performed because of the verification and completion of the results obtained in the Assay 1 was required based on OECD Guideline 476.
In the Assay 2 a 24-h treatment (without S9) was performed according to the ICH recommendation.
A 24-hour treatment was performed without metabolic activation, and a 3-hour treatment with addition of metabolic activation.

Evaluation criteria:

The test item is considered to be mutagenic in this assay if all the following criteria are met (based on Moore et al.):
1. The assay is valid;
2. Statistically significant (p < 0.05) increases in mutation frequency are observed in treated cultures compared to the corresponding negative control values at one or more concentrations;
3. The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
4. There is a significant dose-relationship as indicated by the adequate trend analysis;
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value.

Statistics:

The heterogeneity of the obtained data was tested. The statistical significance of mutant frequencies (total wells with clones) was carried out using Dunnett’s Test, using TOXSTAT statistical software. The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

Results of the Assay 1.:
The treatment duration was 3h and the treatment was performed in presence and also in absence of exogenous metabolic activation (S9 Mix).
In this assay the plating efficiencies of the negative and positive controls in the viability test (PEviability) as well as the mutation frequency of the current negative control were within the acceptable ranges and in accordance with historical data. The parallel tested positive control chemicals induced a statistically significant increase in the mutant frequency (2 Sample t-Test, α = 0.01) additionally the IMF (Induced Mutation Frequency: number of mutants over the control value per 10exp6 viable cells) exceeded the GEF (Global Evaluation Factor, which is equal to 126).
In this assay no cytotoxicity [based on the harmonised relative survival (harmonised RS) and RTG data] was observed at the applied test item concentrations in the absence of S9 Mix, and 65.8 % toxicity (based on the harmonised RS data) was obtained at the concentration level of 1800 μg/mL in presence of S9 Mix.
The evaluated mutation frequencies obtained in the First Assay (Assay 1) did not show any significant differences from the mutation frequency of the corresponding vehicle control and did not show dose-response relationship at the applied concentrations (Dunnett’s Test, α = 0.05). Additionally, the MF values of test item treatments were far below the GEF criterion for positive call.
Precipitation of the test item was not observed during the treatments (either in the presence and absence of S9 Mix).

Results of the Assay 2.:
The treatment duration was 3h in presence of metabolic activation (S9 Mix) and 24h in absence of metabolic activation (S9 Mix).
In the Assay 2 the plating efficiencies of the negative and positive controls in the viability test (PEviability) as well as the mutation frequency of the current negative control were within the acceptable ranges and in accordance with historical data. The parallel tested positive control chemicals induced a statistically significant increase (high above the GEF) in the mutant frequency (2 Sample t-Test, α = 0.01).
3h treatment in presence of S9 Mix:
At the highest examined concentration level of 3000 μg/mL strong cytotoxic effect of the test item was observed.
In this part of the Assay 2 the obtained mutation frequencies were statistically evaluated at the concentration range of 96-2000 μg/mL. The mutation frequency of the concentration level of 2000 μg/mL was statistically significantly higher than the mutation frequency of the vehicle control (Dunnett’s Test, α = 0.05). A slight dose-relationship was also observed between the variances. The higher value remained below the GEF criterion for positive call (IMF: 98), furthermore at this concentration high level of toxicity (89.12 %) was observed.
The obtained statistical significance was not considered as a sign of the mutagenic effect of the test item.
24h treatment in absence of S9 Mix:
The concentration level of 400 μg/mL was very toxic, resulted 2.39 % relative survival (based on the harmonised RS). 12.81 % relative survival was achieved at the concentration level of 280 μg/mL. The relative survival data at the concentration range of 25.6- 160 μg/mL changed in the range of the biological variability of the test.
The mutation frequencies at the concentration levels of 280 and 160 μg/mL were statistically significantly higher than the mutation frequency of the vehicle control (Dunnett’s Test, α = 0.05). The mutation frequencies showed clear dose-related increase, that would confirm a putative potential mutagenic effect of the test item, but similarly to the results of the 3-hour treatments the mutation frequencies remained below the GEF criterion (IMF: 114 at the concentration level of 280 μg/mL and 40 at the concentration level of 160 μg/mL).

Summary of the overall study:
In all phases of the Assay 2 there were one or two concentration levels where the obtained mutation frequencies were statistically significantly higher than the mutation frequencies of the corresponding vehicle control (Dunnett’s Test, α = 0.05). However, the changes of the mutation frequencies were not dose-related, and the GEF criterion for positive call was not attained in any case. In the case of the 3-hour treatments (in absence of S9 at the Completed Assay 1) the higher frequencies were observed in the repeated (partially completed) assays only. In the Assay 1 no statistical significant differences were detected between the treatment and vehicle control mutation rates. Thus, the observed statistical significances were not evaluated as biologically relevant.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the conditions of this study, the test item tetrahydromethylphthalic anhydride (MTHPA) does not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of tetrahydromethylphthalic anhydride (MTHPA) to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (S9 Mix) and for24 hours without metabolic activation.

Exposure concentrations and Vehicle

The test item solutions were prepared in (anhydrous) DMSO and diluted prior to treatment. This vehicle was compatible with the survival of the cells and the S9 activity.

The following concentrations were investigated in the Assay 1:

3-hour treatment (-S9 Mix): 5.12; 12.8; 32; 80; 140 and 200 μg/mL.

3-hour treatment (+S9 Mix): 38.4; 96; 240; 600; 1200 and 1800 μg/mL.

For valid assays the completion of the above concentration series was required. The concentration levels in the Completed Assay 1 were:

3-hour treatment (-S9 Mix): 80; 140; 200; 400; 800 and 1200 μg/mL.

Concentrations were investigated in the Assay 2:

3-hour treatment (+S9 Mix): 96; 240; 600; 1200; 2000 and 3000 μg/mL.

24-hour treatment (-S9 Mix): 25.6; 64; 160; 280; 400 and 800 μg/mL.

Results and Conclusion

In the performed mutation assays, the cell cultures were treated with a range of the test item concentrations. The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments. In the Assay 1, in the absence of S9 Mix no toxic effect of the test item was observed, the changes of the harmonised relative survival and RTG values were in the ranges of the biological variability of the test system. In the presence of S9 Mix 34.22 % harmonised relative survival was observed at the highest examined concentration level, at 1800 μg/mL. The obtained mutation frequencies did not show any statistical or biological significant differences from the negative control values (Dunnett’s Test, α = 0.05). In all phases of the Assay 2 there were one or two concentration levels where the obtained mutation frequencies statistically significantly higher than the mutation frequencies of the corresponding vehicle control (Dunnett’s Test, α = 0.05). However, the changes of the mutation frequencies were dose-related, but the GEF criterion for positive call was not attained in any case. In the case of the 3-hour treatments (in absence of S9) the higher frequencies were observed in the repeated (partially completed) assays only. In the Assay 1 no statistical significant differences were detected between the treatment and vehicle control mutation rates. Thus, the obtained statistical significances were regarded as not biologically relevant.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-2 to 2010-1-4

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

no details given

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: provided by ECACC (European Collection of Cells Cultures)
- Properly maintained: yes, -80 +/- 10°C
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time without and with S9 mix: 19.53, 39.06, 78.12, and 156.25 μg/mL test item.
Experiment B with 20/20 h treatment/sampling time without S9 mix: 1.22, 2.44, 4.88, 9.76 and 19.53 μg/mL test item.
Experiment B with 20/28 h treatment/sampling time without S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL test item.
Experiment B with 3/28 h treatment/sampling time with S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL test item.

The cytotoxicity at the highest concentrations was adequate in the studies (experiment A and experiment B) as indicated by a reduction of % cell survival of at least 50 %.

Vehicle / solvent:

solvent: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethane sulphonate (0.4 and 1.0 μL/mL) and N-Nitrosodimethylamine (1.0 μL/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Experiment A with 3/20 h treatment/sampling time without and with S9 mix: 19.53, 39.06, 78.12, and 156.25 μg/mL test item.
Experiment B with 20/20 h treatment/sampling time without S9 mix: 1.22, 2.44, 4.88, 9.76 and 19.53 μg/mL test item.
Experiment B with 20/28 h treatment/sampling time without S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL test item.
Experiment B with 3/28 h treatment/sampling time with S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL test item.

Preparation of cultures
Cell cultures were treated with Colchicine (0.2 μg/mL) two hours prior to harvest. Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies. For control of bias, all slides were coded and scored blind.

Evaluation criteria:

The Chromosome Aberration Assay is considered valid if following criteria are met:
– the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data (refer to Table 12, "Any other information on results incl. tables").
– the positive control items produce biologically relevant increases in the number of cells with structural chromosome aberrations.

Statistics:

no details given

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment A: 3/20 h: at 156.25 µg/mL (47% rel. surival); Experiment B: 20/20 h: at 19.53 µg/mL (49% rel. surival); Experiment B: 20/28 h: at 78.12 µg/mL (49% rel. surival); Experiment B: 3/28 h: at 78.12 µg/mL (48% rel. surival)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no

EXPERIMENTAL PRE-TEST ON TOXICITY:
The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Chromosome Aberration Assays, both in the absence and in the presence of a metabolic activation system (rodent S9 mix). Toxicity was determined by cell counting and results noted as cell survival in the treatment group (in %) in relation to the negative solvent control. Detailed results of the cytotoxicity assay with the test item are presented in Tables 1 and 2 (refer to "Any other information on results incl. tables"). These results were used to select concentrations of the test item for the Chromosome Aberration Assays.
The following concentrations were selected ranging from little to maximum(< 50% survival) toxicity and evaluated in the main studies (Experiment A and B). All concentrations were run in duplicate (incl. negative and positive controls) and at least 200 well-spread metaphases were assessed:
Experiment A with 3/20 h treatment/sampling time without and with S9 mix: 19.53, 39.06, 78.12, and 156.25 μg/mL test item.
Experiment B with 20/20 h treatment/sampling time without S9 mix: 1.22, 2.44, 4.88, 9.76 and 19.53 μg/mL test item.
Experiment B with 20/28 h treatment/sampling time without S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL test item.
Experiment B with 3/28 h treatment/sampling time with S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL test item.

EXPERIMENTAL PRE-TEST ON TOXICITY

 

Table 1: Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

Test group	Dose µg/mL	S9-mix	Treatment/ sampling time	First count	Second count	Mean value x 10^4 /mL	Relative^a survival in percent
	–	–	3/20	83	83	83	106
	–	–	3/20	80	76	78	100
Test item	2.44	–	3/20	77	81	79	101
	4.88	–	3/20	79	75	77	99
	9.76	–	3/20	77	79	78	100
	19.53	–	3/20	70	72	71	91
	39.06	–	3/20	60	62	61	78
	78.12	–	3/20	52	54	53	68
	156.25	–	3/20	38	36	37	47
	312.50	–	3/20	25	23	24	31
	–	+	3/20	83	83	83	104
	–	+	3/20	81	79	80	100
Test item	2.44	+	3/20	80	76	78	98
	4.88	+	3/20	77	77	77	96
	9.76	+	3/20	76	74	75	94
	19.53	+	3/20	73	71	72	90
	39.06	+	3/20	58	62	60	75
	78.12	+	3/20	53	47	50	63
	156.25	+	3/20	35	37	36	45
	312.50	+	3/20	30	28	29	36

^a Relative to Solvent control

 

Table 2: Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

Test group	Dose µg/mL	S9-mix	Treatment/ sampling time	First count	Second count	Mean value x 10^4 /mL	Relative^a survival in percent
	–	–	20/20	79	83	81	101
	–	–	20/20	79	81	80	100
Test item	2.44	–	20/20	64	66	65	81
	4.88	–	20/20	55	53	54	68
	9.76	–	20/20	45	47	46	58
	19.53	–	20/20	40	38	39	49
	39.06	–	20/20	33	35	34	43
	78.12	–	20/20	25	27	26	33
	156.25	–	20/20	26	24	25	31
	312.50	–	20/20	22	20	21	26

^a Relative to Solvent control

 

Table 2 continued: Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

Test group	Dose µg/mL	S9-mix	Treatment/ sampling time	First count	Second count	Mean value x 10^4 /mL	Relative^a survival in percent
	–	–	20/28	92	90	91	118
	–	–	20/28	78	76	77	100
Test item	2.44	–	20/28	72	72	72	94
	4.88	–	20/28	72	70	71	92
	9.76	–	20/28	59	61	60	78
	19.53	–	20/28	59	57	58	75
	39.06	–	20/28	52	54	53	69
	78.12	–	20/28	39	37	38	49
	156.25	–	20/28	39	33	36	47
	312.50	–	20/28	30	32	31	40
	–	+	3/28	105	109	107	103
	–	+	3/28	103	105	104	100
Test item	2.44	+	3/28	106	102	104	100
	4.88	+	3/28	96	98	97	93
	9.76	+	3/28	85	89	87	84
	19.53	+	3/28	74	72	73	70
	39.06	+	3/28	65	61	63	61
	78.12	+	3/28	49	51	50	48
	156.25	+	3/28	44	44	44	42
	312.50	+	3/28	40	38	39	38

^a Relative to Solvent control

 

 

HISTORICAL CONTROL DATA

 

TABLE 12:

3/20h treatment/sampling time without S9-mix

	aberration rate (phases with aberration in %)
	negative control		positive control (EMS)
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
Mean	3.31	1.25	26.00	22.81
SD.	1.08	0.77	3.52	3.60
Range	2-6	0-3	20-33	18-31
n	8	8	8	8

 

3h/20h treatment/sampling time with S9-mix

	aberration rate (phases with aberration in %)
	negative control		positive control (NNDA)
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
Mean	3.00	1.13	21.50	18.81
SD.	0.89	0.62	2.03	1.47
Range	2-5	0-2	17-26	16-21
n	8	8	8	8

 

20h/28h treatment/sampling time without S9-mix

	aberration rate (phases with aberration in %)
	negative control		positive control (EMS)
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
Mean	3.38	1.56	27.94	25.25
SD.	0.72	0.51	3.32	3.44
Range	2-5	1-2	22-35	20-31
n	8	8	8	8

 

3h/28h treatment/sampling time without S9-mix

	aberration rate (phases with aberration in %)
	negative control		positive control (NNDA)
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
Mean	3.38	1.63	20.75	18.88
SD.	0.96	0.72	1.98	2.06
Range	2-5	0-3	18-26	16-24
n	8	8	8	8

 

SD         =  standard deviation

Range  =  min.-max. values

n            =  number of experiments

EMS     =  Ethyl methanesulphonate

NNDA  =  N-Nitrosodimethylamine

 

Please refer to section "'Overall remarks, attachments" for further experimental tables.

Conclusions:

In conclusion, tetrahydromethylphthalic anhydride (MTHPA) tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, tetrahydromethylphthalic anhydride (MTHPA) is considered as not clastogenic in this system.

Executive summary:

The test item, tetrahydromethylphthalic anhydride (MTHPA) was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation).

In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (< 50 % survival) toxicity:

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix: 19.53, 39.06, 78.12, and 156.25 μg/mL test item.

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 1.22, 2.44, 4.88, 9.76 and 19.53 μg/mL test item.

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL test item.

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL test item.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent control, when tetrahydromethylphthalic anhydride (MTHPA) was examined up to cytotoxic concentrations (1.22 2.44, 4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL) without S9 mix over a prolonged treatment period 20 hour treatment and 20 and 28 hour sampling times. Further, a three-hour treatment with tetrahydromethylphthalic anhydride (MTHPA) up to cytotoxic concentrations (4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL) in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps.

As in Experiment A, in Experiment B no statistically significant differences between treatment and control groups and no dose-response relationships were noted.

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The validity of the test was shown using Ethylmethane sulphonate (0.4 and 1.0 μL/mL) and N-Nitrosodimethylamine (1.0 μL/mL) as positive controls.

In conclusion, tetrahydromethylphthalic anhydride (MTHPA) tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, tetrahydromethylphthalic anhydride (MTHPA) is considered as not clastogenic in this system. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

A reverse gene mutation assay was conducted in line with Guidance for Screening Mutagenicity Testing of Chemicals (Japan) and OECD Test Guidelines 471 and 472, using the pre-incubation method. Therefore it was identified as a key study.

In a reverse gene mutation assay in bacteria, strains TA100, TA1535, TA98, TA1537 of S. typhimurium and Escherichia coli Wp2 uvrA were exposed to tetrahydromethylphthalic anhydride in DMSO at concentrations of 0, 62.5, 125, 250, 500, 1000, 2000 ug/plate (-S9 mix) and 0, 156, 313, 625, 1250, 2500, 5000 ug/plate (+S9 mix) in the presence and absence of mammalian metabolic activation (S9 mix, Rat liver, induced with Phenobarbital and 5,6-benzoflavone).

Tetrahydromethylphthalic anhydride was tested up to limit concentration (5000 µg/plate). No cytotoxicity occurred. The test chemical did not induce mutations in the S. typhimurium and E. coli strains The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for the Japanese Guidelines for Screening Mutagenicity Testing of Chemicals Test Guideline and OECD 471 and 472 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

 

Chromosome Aberration tests:

Two chromosome aberrration tests were conducted with the test substance tetrahydromethylphthalic anhydride (MTHPA).

 

In a first mammalian cell cytogenetics assay (supporting, Tanaka, 1997), CHL/IU cell cultures/primary lymphocyte cultures were exposed tetrahydromethylphthalic anhydride (MTHPA, CAS 11070-44-3) at concentrations of 0, 0.075, 0.15, 0.30 and 0.60 mg/ml with and without metabolic activation (S9).

Structural chromosomal aberrations were not induced up to 0.30 mg/ml (24 and 48hr continuous treatment without S9). Polyploidy (1.13 %) was increased at 0.30 mg/ml with 48 hr continuous treatment without metabolic activation. Furthermore, polyploidy (1.25-1.88 %) was statistically increased at 0.11-0.43 mg/ml (all concentrations) with short-term treatment with an exogenous metabolic activation system, indicating a potential effect of the test item. Whereas this study showed no indication of clastogenic properties, a polyploidy inducing effect cannot be excluded. Therefore a second test was performed. However, the biological relevance of the observed variation was considered equivocal as the test design is of limited use to investigate such effects.

 

In the key study (Beres, 2010), the test item, tetrahydromethylphthalic anhydride (MTHPA) was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation).

In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (< 50 % survival) toxicity:

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix: 19.53, 39.06, 78.12, and 156.25 μg/ml test item.

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 1.22, 2.44, 4.88, 9.76 and 19.53 μg/ml test item.

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/ml test item.

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/ml test item.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent control, when tetrahydromethylphthalic anhydride (MTHPA) was examined up to cytotoxic concentrations (1.22 2.44, 4.88, 9.76, 19.53, 39.06 and 78.12 μg/ml) without S9 mix over a prolonged treatment period of 20 hour and 20 and 28 hour sampling times. Further, a three-hour treatment with tetrahydromethylphthalic anhydride (MTHPA) up to cytotoxic concentrations (4.88, 9.76, 19.53, 39.06 and 78.12 μg/ml) in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps.

As in Experiment A, in Experiment B no statistically significant differences between treatment and control groups and no dose-response relationships were noted.

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The validity of the test was shown using Ethylmethane sulphonate (0.4 and1.0 μl/ml) and N-Nitrosodimethylamine (1.0 μl/ml) as positive controls.

In conclusion, tetrahydromethylphthalic anhydride (MTHPA) tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells and no indications of polyploidy were noted. Therefore, tetrahydromethylphthalic anhydride (MTHPA) is considered as not clastogenic in this system. 

 

Mouse Lymphoma assay:

In the performed mutation assays (Vertesi, 2009), the cell cultures were treated with a range of tetrahydromethylphthalic anhydride (MTHPA) concentrations. The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments. In the Assay 1, in the absence of S9 Mix no toxic effect of the test item was observed, the changes of the harmonised relative survival and RTG values were in the ranges of the biological variability of the test system. In the presence of S9 Mix 34.22 % harmonised relative survival was observed at the highest examined concentration level, at 1800 μg/mL. The obtained mutation frequencies did not show any statistical or biological significant differences from the negative control values (Dunnett’s Test, α = 0.05). In all phases of the Assay 2 there were one or two concentration levels where the obtained mutation frequencies statistically significantly higher than the mutation frequencies of the corresponding vehicle control (Dunnett’s Test, α = 0.05). However, the changes of the mutation frequencies were dose-related, but the GEF criterion for positive call was not attained in any case. In the case of the 3-hour treatments (in absence of S9) the higher frequencies were observed in the repeated (partially completed) assays only. In the Assay 1 no statistical significant differences were detected between the treatment and vehicle control mutation rates. Thus, the obtained statistical significances were regarded as not biologically relevant.

Genetic toxicity was evaluated taking into account an Ames test, a MLA and 2 CA tests. Based on these studies and their experimental outcomes, MTHPA was concluded to be negative in regard to mutagenicity and chromosomal aberration.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.