Phthalimide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test OECD TG 471: S. typhimurium TA 98,100,1535,1537; E.coli WP2 uvrA 

negative with and without metabolic activation

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operone

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 from male rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

0, 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9-mix: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (TA98, TA100), sodium azide (TA1535), 9-aminoacridine (TA1537), N-ethyl-N'-nitro-N-nitrosoguanidine (WP2); with S9-mix; 2-aminoanthracene (all five strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
NUMBER OF REPLICATIONS: 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No cytotoxicity was observed up to 5000 µg/plate, the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No cytotoxicity was observed up to 5000 µg/plate, the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No cytotoxicity was observed up to 5000 µg/plate, the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No cytotoxicity was observed up to 5000 µg/plate, the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No cytotoxicity was observed up to 5000 µg/plate, the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Phthalimide did not induce mutations in the S. typhimurium and E. coli strains. No toxicity was observed up to a concentration of 5000 µg/plate, with or without metabolic activation. The positive controls had a marked mutagenic effect, as was seen by a relevant increase in mutant colonies compared to the corresponding negative controls

Table 1: Results of reverse mutation test of phthalimide on bacteria (I)

 

With or without S9-Mix	Test substance concentration (µg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
-	0	162 ± 12	8 ± 2	28 ± 2	16 ± 6	8 ± 3
-	313	158 ± 7	8 ± 3	28 ± 8	21 ± 4	7 ± 2
-	625	161 ± 24	10 ± 3	33 ± 3	18 ± 3	7 ± 2
-	1250	159 ± 7	11 ± 4	29 ± 3	19 ± 3	9 ± 1
-	2500	168 ± 12	11 ± 1	32 ± 3	15 ± 3	5 ± 1
-	5000	143 ± 9	11 ± 1	27 ± 3	15 ± 2	6 ± 2
Positive controls - S9	Name	AF2	NaN3	ENNG	AF2	9AA
	Concentrations (µg/plate)	0.01	0.5	2	0.1	80
	Number of revertants	531 ± 26	517 ± 22	392 ± 21	372 ± 32	197 ± 20
+	0	187 ± 11	15 ± 4	33 ± 6	27 ± 10	12 ± 1
+	313	188 ± 13	12 ± 3	35 ± 2	30 ± 4	7 ± 2
+	625	164 ± 30	16 ± 1	33 ± 1	30 ± 1	9 ± 2
+	1250	170 ± 20	11 ± 3	33 ± 4	20 ± 1	7 ± 2
+	2500	164 ± 18	9 ± 2	31 ± 8	27 ± 4	8 ± 1
+	5000	154 ± 9	16 ± 1	33 ± 7	27 ± 10	8 ± 2
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (µg/plate)	1	2	10	0.5	2
	Number of revertants	1127±50	390±31	1555±119	299±47	178±36

 

AF2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3=Sodium azide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine

Table 2: Results of reverse mutation test of phthalimide on bacteria (II)

 

With or with out S9-Mix	Test substance concentration (µg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
-	0	132 ± 10	11 ± 2	23 ±7	19 ± 6	8 ± 2
-	313	120 ± 8	11 ± 2	29 ±7	17 ± 2	7 ± 3
-	625	119 ± 6	9 ± 1	27 ±1	18 ± 3	6 ± 3
-	1250	130 ± 7	9 ± 1	20 ±1	19 ± 2	5 ± 2
-	2500	122 ±12	9 ± 1	19 ±2	23 ± 6	8 ± 2
-	5000	105 ± 5	10±2	22 ±5	15 ± 3	8 ± 3
Positive controls - S9	Name	AF2	NaN3	ENNG	AF2	9AA
	Concentrations (µg/plate)	0.01	0.5	2	0.1	80
	Number of revertants	462 ± 13	525 ± 15	630 ± 8	355 ± 20	246 ± 34
+	0	144 ± 13	13 ± 6	27 ± 2	32 ± 3	13 ± 5
+	313	151 ± 15	19 ± 4	28 ± 2	24 ± 4	9 ± 2
+	625	154 ± 5	10 ± 2	27 ± 5	35 ± 7	13 ± 3
+	1250	142 ± 17	16 ± 5	30 ± 7	30 ± 6	11 ± 1
+	2500	139 ± 12	14 ± 3	26 ± 6	28 ± 5	8 ± 3
+	5000	138 ± 12	9 ± 1	24 ± 2	28 ± 7	9 ± 1
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (µg/plate)	1	2	10	0.5	2
	Number of revertants	1033 ± 77	297 ± 32	1246 ± 70	341 ± 4	145 ±16

 

AF2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3=Sodium azide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine

Conclusions:

negative with metabolic activation
negative without metabolic activation

Executive summary:

MHW Japan (1999):

Salmonella typhimurium strains TA100, TA1535, TA98, TA1537, and E.coli WP2 uvrA were treated with the test material diluted in DMSO using the Ames plate incorporation method according to OECD Guideline No. 471 at five dose levels, in duplicate, both with and without the addition of a rat liver homogenate metabolising system. The dose range was from 313 to 5000 µg/plate for both of two consecutive experiments. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Phthalimide is not active in a variety of mutagenicity tests in vitro. It is not mutagenic in bacterial reverse mutation test (OECD TG 471) in the presence and absence of metabolic activation system (S9-mix). It does not induce mutations in the mouse lymphoma assay. In the chromosomal aberration test (OECD TG 473) no polyploidy is observed at any concentration in the absence and presence of metabolic activation. Weak clastogenic effects are observed in the presence of metabolic activation at high concentrations where cytotoxicity is seen in parallel. Genotoxicity studies in vivo are not available. Overall phthalimide is considered to be not genotoxic in vitro and based on the available data it is anticipated that it will not be genotoxic in vivo.

Short description of key information:
OECD SIDS
Studies in Animals
In vitro Studies
Phthalimide was not mutagenic in bacterial reverse mutation tests (OECD TG 471 and 472) in the absence or presence of metabolic activation in different Salmonella typhimurium and Escherichia coli strains at doses of up to 5000 µg per plate by the pre-incubation or plating method (Bayer AG, 1993; MHW Japan, 1999).
In a chromosomal aberration test (OECD TG 473) no polyploidy or clastogenicity was observed in the absence of metabolic activation in Chinese hamster CHL/IU cells. Slight induction of structural chromosome aberrations was observed in the presence of metabolic activation at the 2500 µg/ml dose, and this observation became statistically significant at 5000 µg/ml. This weak induction was observed only at high concentrations where cytotoxicity was seen in parallel (50% growth inhibition concentration with S9-mix 4524 µg/mL) (MHW Japan, 1999). In view that the observed effects just meets the investigator´s criterion for a positive response (>= 10%) and the effects were seen only at high, cytotoxic concentrations the relevance of these observations appears to be rather limited. Consequently, phthalimide was considered to be not clastogenic (MHW Japan, 1999).
Positive and negative controls gave the expected results in all in vitro Studies reported.
In vivo Studies
No data on in vivo mutagenicity are available

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data there is no reason for classification.