4-isopropylaniline

Genetic toxicity: in vitro

Administrative data

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:

December 20th, 1988 - February 9th, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

: no historical data reported

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-isopropylaniline

EC Number:

202-797-2

EC Name:

4-isopropylaniline

Cas Number:

99-88-7

Molecular formula:

C9H13N

IUPAC Name:

4-(propan-2-yl)aniline

Details on test material:

- Name of test material (as cited in study report): Cumidin
- Chemical name: 1-Amino-4-isopropylbenzol
- Physical state: darkbrown liquid
- Analytical purity: > 98 %
- Code: GKGB 155
- Charge No.: 55. Kamp.
- Expiration date of the lot/batch: not specified by the sponsor
- Storage condition of test material: darkness at 20 degree C

Method

Target gene:

HGPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM with Hanks-salts and 25 mM Hepes-buffer
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

0, 50, 200, 350 and 500 µg/mL
(at 600 and 700 µg /mL because of high cytotoxicity no mutant selection was performed)

Vehicle / solvent:

At the day of the experiment the test substance was dissolved in ethanol (final concentration max 1 %).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

the final ethanol concentration did not exceed 1 %

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: ethylmethanesulphonate; with metabolic activation: 9,10-dimethylbenzanthracene in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 4 hours of treatment with test substance with and without S9-mix
- Expression time (cells in growth medium): 7 days (2 subcultures were made)
- Selection time (if incubation with a selection agent): 7 days in medium containing 6-thioguanine
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days after the 4h-exposure the cells were fixed and stained with 10% methylene blue in 0.01 n KOH solution

SELECTION AGENT (mutation assays): Thioguanine (0.11 % in the medium)

NUMBER OF REPLICATIONS: Two independent experiments with and without S9 -mix were performed.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

OTHER: RATIONALE FOR DOSE SELECTION
The dose level which resulted in a 30 % survival in the preliminary cytotoxicity experiment was chosen as the highest dose level.
The lowest doses were chosen based on the level from the negative controls.

Evaluation criteria:

Mutagenic,
- if the mutation frequency was 3 times higher than the spontaneous frequency in this experimental setting and
- if the mutation frequency was increased in a concentration - related manner.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 750 µg/mL and above. No precipitation was visible at the highest soluble concentration of 1352 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: up to highest soluble concentrations no precipitation observed.

RANGE-FINDING/SCREENING STUDIES:
500 µg/mL was determined in the absence and presence of metabolic activation as the maximal dose level for mutagenicity testing. Higher doses showed toxic effects on the cells.

COMPARISON WITH HISTORICAL CONTROL DATA:
no historical data shown

Any other information on results incl. tables

Table 1: Mutagenicity Data - Experiment I

 

	Dose per mL	S9-mix	Number of mutant colonies					mean	Standard deviation	Mutation frequency*
			I	II	III	IV	V
Negative control	0	-	19	20	15	15	14	16.6	2.70	63.4
Solvent control	0	-	10	15	11	14	6	11.2	3.56	52.3
Positive control: EMS	1.0 mg	-	108	120	118	105	108	111.8	6.72	463.8
Cumidin	50 µg	-	12	10	18	15	24	15.8	5.50	61.1
	200 µg	-	13	26	22	18	16	19.0	5.10	81.0
	350 µg	-	8	6	6	16	16	10.4	5.18	44.9
	500 µg	-	12	12	10	11	9	10.8	1.30	58.7
Negative control	0	+	18	17	22	11	13	16.2	4.32	56.9
Solvent control	0	+	17	15	10	16	18	15.2	3.11	46.9
Positive control: DMBA	7.7 µg	+	156	187	164	194	150	170.2	19.34	890.7
Cumidin	50 µg	+	20	17	20	12	12	16.2	4.01	76.0
	200 µg	+	10	11	14	15	16	13.2	2.59	53.0
	350 µg	+	9	8	8	9	12	9.2	1.64	50.1
	500 µg	+	13	6	3	6	5	6.6	3.78	26.9

*Mutation frequency (mutanta colonies per 10⁶cells): mean value x 10⁶/ cells survived (data given in original report)

 

Table 2: Mutagenicity Data - Experiment II

 

	Dose per mL	S9-mix	Number of mutant colonies					mean	Standard deviation	Mutation frequency*
			I	II	III	IV	V
Negative control	0	-	15	9	15	5	11	11.0	4.24	59.2
Solvent control	0	-	16	16	15	16	29	18.4	5.94	94.8
Positive control: EMS	1.0 mg	-	89	110	85	116	125	105.0	17.33	477.4
Cumidin	50 µg	-	12	12	15	18	12	13.8	2.68	63.7
	200 µg	-	9	15	14	17	15	14.0	3.00	79.0
	350 µg	-	10	6	6	14	16	10.4	4.56	77.1
	500 µg	-	7	6	7	11	7	7.6	1.95	40.8
Negative control	0	+	5	10	14	13	14	11.2	3.83	39.8
Solvent control	0	+	14	15	15	15	10	13.8	2.17	52.9
Positive control: DMBA	7.7 µg	+	112	99	109	132	126	115.4	13.63	671.3
Cumidin	50 µg	+	22	13	22	16	17	18.0	3.94	121.7
	200 µg	+	9	18	15	7	11	12.0	4.47	78.1
	350 µg	+	11	10	11	13	11	11.2	1.10	58.3
	500 µg	+	25	14	10	24	19	18.4	6.43	98.2

*Mutation frequency (mutanta colonies per 10⁶cells): mean value x 10⁶/ cells survived (data given in original report)

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative in HGPRT-test with cells of the V79 Chinese hamster cell line.

Executive summary:

An in vitro mammalian cell gene mutation assay was performed according to OECD guideline 476. The mutagenic potential of Cumidin was tested using the method for induction of the HPRT gene in V79 Chinese hamster cells at dose levels of 0, 50, 200, 350 and 500 µg /mL in two independent experiments with and without metabolic activation (S9 -mix). In a preliminary cytotoxicity experiment significant cytotoxic effects were observed at concentrations ranging from 750 - 1352 µg /mL.

The test compound did not induce a significant increase in the number of mutant colonies in comparison to negative and positive controls. In conclusion, the test substance Cumidin did not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, either with or without metabolic activation under the described experimental conditions.