Reaction mass of 2-hydroxyisopropyl 2-hydroxypropyl bicarbamate and 2-hydroxypropyl carbazate and propylene carbonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Mar to May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-hydroxyisopropyl 2-hydroxypropyl bicarbamate and 2-hydroxypropyl carbazate and propylene carbonate

EC Number:

906-521-8

Cas Number:

85391-60-2

Molecular formula:

C8H16N2O6

IUPAC Name:

Reaction mass of 2-hydroxyisopropyl 2-hydroxypropyl bicarbamate and 2-hydroxypropyl carbazate and propylene carbonate

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: P2D5000133
- Purity: 100 %
- Expiration date of the lot/batch: 2015-06-12

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

0, 50, 160, 500, 1600, 5000 µg/plate (without and with S9 mix)

Vehicle / solvent:

Solvents used: demineralized water (test substance), deionized water (mitomycin C), DMSO (sodium azide, cumene hydroperoxide, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, 2-aminoanthracene)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 100, TA 1535), 2-nitrofluorene (TA 98), 4-nitro-1,2-phenylene diamine (TA 1537), mitomycin C (TA 102, plate incorp.), cumene hyd (TA 102, preincub. trials), 2-aminoanthracene (all strains)

Remarks:

The positive controls sodium azide, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, mitomycin C and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.

Details on test system and experimental conditions:

METHOD: Standard plate test and preincubation test

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 1537, TA 100 and TA 98 this increase should be about twice that of negative controls. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

not specified

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 102, TA 1535, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 102, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: plate incorporation test

Any other information on results incl. tables

Table 1: Summary of results from the Salmonella mutagenicity assay (plate incorporation test) with KGD 1409 (mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (demin. water)	13	110	5	16	205
50	10	104	4	16	210
160	10	91	6	12	218
500	9	97	5	14	200
1600	10	92	6	15	201
5000	15	111	4	18	203
Positive control	660	1395	39	1167	782
Dose (µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (demin. water)	12	154	9	28	296
50	11	139	9	29	329
160	11	164	10	26	321
500	13	134	8	31	301
1600	15	148	8	25	300
5000	20	144	8	26	272
Positive control	132	2980	279	2791	1158

Table 2: Summary of results from the Salmonella mutagenicity assay (independent preincubation test) with KGD 1409 (mean values of revertants per plate)

 

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (demin. water)	10	114	6	16	240
50	10	114	7	21	245
160	8	111	7	20	261
500	10	105	6	13	253
1600	8	109	7	16	259
5000	20	118	8	17	265
Positive control	656	842	37	1101	550
Dose (µg per plate)	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (demin. water)	10	156	10	27	334
50	10	150	8	28	343
160	11	162	9	27	341
500	9	164	9	28	339
1600	15	162	8	26	359
5000	25	178	8	28	337
Positive control	239	2204	382	2251	1013

 

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects or precipitations.

Evidence of mutagenic activity of the test item was seen. On Salmonella typhimurium TA1535, a biologically relevant increase was found in the mutant count compared to the corresponding solvent control when using the preincubation method. Both with and without S9 mix, there was a positive response. The lowest reproducible effective dose was 5000 µg per plate for Salmonella typhimurium TA1535. The Salmonella/microsome test thus showed the test item to have a mutagenic effect.

In both experiments the positive controls sodium azide, 4-nitro-1,2-phenylene diamine, 2-nitrofluoren, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Applicant's summary and conclusion

Conclusions:

positive

Executive summary:

The mutagenic potential of KGD 1409 was evaluated in a Salmonella/microsome test (plate incorporation test and preincubation methood) with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471.

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects or precipitations.

Evidence of mutagenic activity of the test item was seen. On Salmonella typhimurium TA1535, a biologically relevant increase was found in the mutant count compared to the corresponding solvent control when using the preincubation method. Both with and without S9 mix, there was a positive response. The lowest reproducible effective dose was 5000 µg per plate for Salmonella typhimurium TA1535.

Therefore, the test item was considered to be mutagenic without and with S9 mix in the preincubation modification of the Salmonella/microsome test.