3-hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2-dimethylpropionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 10, 2023 - Jan 26, 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 Jun 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Rheinland Pfalz, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2-dimethylpropionate

EC Number:

214-222-2

EC Name:

3-hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2-dimethylpropionate

Cas Number:

1115-20-4

Molecular formula:

C10H20O4

IUPAC Name:

3-hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2-dimethylpropanoate

Details on test material:

- Name of test material (as cited in study report): Hydroxypivalic acid neopentylglycol ester
- Batch identification: 09129047G0
- Purity: 99.6 %
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
- Stability under test conditions: The stability of the test substance under storage conditions was guaranteed until 04 Apr 2024 as indicated by the sponsor and the sponsor holds this responsibility.The test facility is organizationally independent from the BASF SE sponsor division.
- Date of production: 05 Apr 2022
- Physical state, appearance: solid, white
- Storage conditions: room temperature

Method

Target gene:

his, trp

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: rat liver S9-mix
- Source of S9: male Wistar rats i.p. injected with 80 mg/kg b.w. phenobarbital and ß-naphthoflavone orally each on three consecutive days; 24 h after the last administration rats were sacrificed and liver S9-fraction prepared
- Method of preparation of S9 mix: was prepared freshly prior to each experiment; a sufficient amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors: MgCl2 8mM, KCl 33 mM, glucose-6-phosphate 5 mM, NADP 4 mM, phosphate buffer pH 7.4 15mM); mixture of both components (S9 mix) was kept on ice until used
- Concentration or volume of S9 mix and S9 in the final culture medium
- Quality controls of S9: sterility of the S9 fraction was assessed and confirmed; to demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene

Test concentrations with justification for top dose:

33 μg - 5000 μg/plate (SPT)
33 μg - 5000 μg/plate (PIT)

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate is generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose can be tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation. In this study, 5 mg/plate could be used as top concentration.

Vehicle / solvent:

Due to the good solubility of the test substance in water, ultrapure water was used as vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA), N-methyl-N`-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD),

Details on test system and experimental conditions:

MUTAGENICITY TESTS
i) Standard plate test (3 test plates per dose or per control)
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al. (1975, 1983).
a) Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order: 0.1 mL test solution, vehicle or positive control, 0.1 mL fresh bacterial culture, 0.5 mL S9 mix (with external metabolic activation) or 0.5 mL phosphate buffer (without external metabolic activation). After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds. After incubation at 37°C (mean value ±2°C) for 48-72 h in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. In several cases, colonies were counted manually, in particular, if precipitation of the test substance hindered the counting using the Image Analysis System.
b) Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order: 0.1 mL test solution, vehicle or positive control, 0.1 mL fresh bacterial culture, 0.5 mL S9 mix (with external metabolic activation) or 0.5 mL phosphate buffer (without external metabolic activation). After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 sec. After incubation at 37°C (mean value ±2°C) for 48-72 h in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. In several cases, colonies were counted manually, in particular, if precipitation of the test substance hindered the counting using the Image Analysis System.

ii) Preincubation test (3 test plates per dose or per control)
The experimental procedure was based on the method described by Yahagi et al. (1977) and Matsushima et al. (1980). 0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with external metabolic activation) or phosphate buffer (without external metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds. After incubation at 37°C (mean value ±2°C) for 48-72 h in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. In several cases, colonies were counted manually, in particular, if precipitation of the test substance hindered the counting using the Image Analysis System.

EVALUATION OF TOXICITY
Toxicity defined by a decrease in the number of revertants (factor ≤ 0.6) and clearing or diminution of the background lawn (= reduced his- or trp- background growth) was investigated for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 are not detected as toxicity in low dose groups.

EVALUATION OF SOLUBILTY
If precipitation of the test material is observed, it is recorded. As long as precipitation do not interfere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

ACCEPTANCE CRITERIA
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls are within the range of the historical negative control data for each tester strain. If a value is not within the control range, the value might be accepted if it is not a clear outlier and there is evidence that the test system is “under control” and there is evidence of no technical or human failure
- The sterility controls reveal no indication of bacterial contamination. Test substance precipitation should not interfere with the scoring.
- The positive control substances both with and without S9 mix induce a distinct increase in the number of revertant colonies compatible with the range of the historical positive control data or above
- Fresh bacterial culture containing approximately 109 cells per mL are used.

Rationale for test conditions:

In agreement with the recommendations of current guidelines.

Evaluation criteria:

The test substance is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains are within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

not specified

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only in the PIT with S9 mix at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium 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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only in the SPT with S9 mix at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only in the PIT with S9 mix at 5000 µg/plate

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
No test substance precipitation was observed with and without S9 mix.
The additional treated plates for sterility control showed no contamination in all performed experiments.

STUDY RESULTS
No relevant increase in the number of his+ or trp+ revertants with and without S9 mix in all strains tested (see tables 1-4).
A bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in the standard plate test depending on the strain and test conditions at 2500 μg/plate. In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions at 5000 μg/plate.

HISTORICAL CONTROL DATA
see tables 5-8

Any other information on results incl. tables

Table 1: Mutagenicity without metabolic activation - SPT

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water	-	16.3	0.6	-	16, 16, 17
	Test Item	33	15.3	6.5	0.9	15, 22 ,9
		100	17.0	1.0	1.0	17, 16, 18
		333	20.3	2.5	1.2	18, 20, 23
		1000	16.7	3.5	1.0	13, 17, 20
		2500	11.3	3.2	0.7	10, 9, 15
		5000	12.0	5.0	0.7	12, 17, 7
	MNNG	5.0	2356.3	434.5	144.3	2432, 2748, 1889
TA 100	Water	-	135.7	16.3	-	117, 143, 147
	Test Item	33	129.0	10.5	1.0	119, 140, 128
		100	143.3	7.5	1.1	136, 151, 143
		333	136.0	14.7	1.0	128, 153, 127
		1000	133.3	6.4	1.0	138, 126, 136
		2500	147.3	10.5	1.1	147, 137, 158
		5000	146.0	1.0	1.1	146, 145, 147
	MNNG	5.0	2243.0	421.2	16.5	2657, 2257, 1815
TA 1537	Water	-	11.3	3.1	-	8, 14, 12
	Test Item	33	11.0	4.4	1.0	9, 16, 8
		100	11.7	7.6	1.0	17, 15, 3
		333	13.3	4.2	1.2	12, 18, 10
		1000	8.3	3.2	0.7	12, 7, 6
		2500	10.7	5.0	0.9	16, 6, 10
		5000	13.0	7.0	1.1	21, 10, 8
	AAC	100	1005.3	240.0	88.7	775, 987, 1254
TA 98	Water	-	17.7	2.1	-	16, 17, 20
	Test Item	33	26.3	5.5	1.5	30, 29, 20
		100	17.7	4.2	1.0	21, 13, 19
		333	17.3	6.0	1.0	23, 11, 18
		1000	13.7	5.7	0.8	20, 9, 12
		2500	20.0	4.6	1.1	19, 25, 16
		5000	25.7	1.5	1.5	24, 26, 27
	NOPD	10	469.3	109.3	26.6	595, 417, 396
E. coli	Water	-	26.7	3.2	-	23, 29, 28
	Test Item	33	18.3	2.9	0.7	20, 15, 20
		100	25.7	5.5	1.0	32, 22, 23
		333	25.7	0.6	1.0	25, 26, 26
		1000	24.0	2.6	0.9	21, 25, 26
		2500	27.0	3.6	1.0	24, 31, 26
		5000	20.3	5.5	0.8	15, 26, 20
	4-NQO	5	1011.0	41.2	37.9	1041, 964, 1028

 

Table 2: Mutagenicity with metabolic activation - SPT

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water	-	11.3	1.5	-	11, 10, 13
	Test Item	33	8.3	2.9	0.7	10, 10, 5
		100	14.3	2.1	1.3	12, 15, 16
		333	11.3	4.2	1.0	8, 16, 10
		1000	10.7	2.5	0.9	13, 11,8
		2500	8.7	6.0	0.8	8, 15, 3
		5000	10.7	0.6	0.9	11, 10, 11
	MNNG	5.0	162.0	14.8	14.3	179, 152, 155
TA 100	Water	-	133.0	5.3	-	131, 139, 129
	Test Item	33	129.3	7.1	1.0	123, 128, 137
		100	131.7	21.1	1.0	118, 121, 156
		333	131.3	9.7	1.0	123, 129, 142
		1000	137.0	4.0	1.0	133, 137, 141
		2500	122.7	3.1	0.9	120, 126, 122
		5000	122.3	17.8	0.9	138, 103, 126
	MNNG	5.0	1426.0	122.6	10.7	1552, 1419, 1307
TA 1537	Water	-	13.0	3.6	-	17, 10, 12
	Test Item	33	9.3	4.0	0.7	13, 10, 5
		100	12.7	2.5	1.0	10, 15, 13
		333	10.0	2.0	0.8	8, 12, 10
		1000	14.0	5.6	1.1	8, 15, 19
		2500	13.7	5.5	1.1	10, 11, 20
		5000	14.0	1.0	1.1	13, 15, 14
	AAC	100	96.3	2.1	7.4	94, 97, 98
TA 98	Water	-	25.3	3.8	-	21, 28, 27
	Test Item	33	23.3	4.0	0.9	21, 28, 21
		100	16.0	4.4	0.6	13, 21, 14
		333	25.0	4.4	1.0	20, 27, 28
		1000	24.0	4.4	0.9	26, 27, 19
		2500	13.7	2.1	0.5	16, 13, 12
		5000	20.0	3.5	0.8	24, 18, 18
	NOPD	10	872.7	87.1	34.4	934, 911, 773
E. coli	Water	-	27.3	5.7	-	29, 21, 32
	Test Item	33	29.7	2.3	1.1	31, 31, 27
		100	29.3	3.5	1.1	26, 29, 33
		333	27.0	6.2	1.0	25, 22, 34
		1000	20.7	5.5	0.8	26, 15, 21
		2500	31.7	2.1	1.2	30, 31, 34
		5000	23.0	4.6	0.8	27, 24, 18
	4-NQO	5	118.0	10.4	4.3	130, 111, 113

 

Table 3: Mutagenicity without metabolic activation - PIT

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water	-	13.0	1.0	-	13, 14, 12
	Test Item	33	7.7	0.6	0.6	8, 8, 7
		100	11.0	2.6	0.8	9, 14, 10
		333	14.3	2.3	1.1	13, 17, 13
		1000	10.7	3.8	0.8	9, 8, 15
		2500	9.0	1.0	0.7	9, 8, 10
		5000	12.3	2.1	0.9	14, 13, 10
	MNNG	5.0	5219.7	289.6	401.5	5060, 5554, 5045
TA 100	Water	-	127.7	19.6	-	144, 133, 106
	Test Item	33	120.3	7.5	0.9	116, 116, 129
		100	110.3	9.7	0.9	108, 121, 102
		333	123.7	13.5	1.0	137, 124, 110
		1000	123.0	10.4	1.0	117, 117, 135
		2500	120.3	10.7	0.69	108, 127, 126
		5000	113.7	5.9	0.9	107, 118, 116
	MNNG	5.0	4176.0	395.8	32.7	4431, 4377, 3720
TA 1537	Water	-	12.0	2.0	-	14, 12, 10
	Test Item	33	15.7	3.2	1.3	12, 18, 17
		100	12.7	4.9	1.1	15, 16, 7
		333	14.3	4.2	1.2	19, 11, 13
		1000	9.7	1.2	0.8	11, 9, 9
		2500	12.7	5.8	1.1	16, 16, 6
		5000	8.3	3.1	0.7	11, 5, 9
	AAC	100	1249.7	371.3	104.1	1174, 922, 1653
TA 98	Water	-	27.0	6.6	-	34, 21, 26
	Test Item	33	19.7	8.1	0.7	14, 16, 29
		100	20.3	6.7	0.8	13, 26, 22
		333	18.7	5.9	0.7	12, 21, 23
		1000	23.3	7.0	0.9	16, 24, 30
		2500	25.0	7.0	0.9	32, 25, 18
		5000	22.3	2.1	0.8	20, 23, 24
	NOPD	10	395.7	35.1	14.7	359, 399, 429
E. coli	Water	-	26.7	1.2	-	26, 26, 28
	Test Item	33	28.7	3.5	1.1	25, 32, 29
		100	27.3	9.0	1.0	32, 17, 33
		333	25.0	3.6	0.9	29, 24, 22
		1000	24.7	6.0	0.9	24, 19, 31
		2500	18.0	6.1	0.7	15, 14, 25
		5000	17.7	1.5	0.7	16, 18, 19
	4-NQO	5	962.3	6.4	36.1	967, 965, 955

 

Table 4: Mutagenicity with metabolic activation - PIT

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water	-	16.0	7.0	-	11, 24, 13
	Test Item	33	9.3	1.5	0.6	9, 11, 8
		100	10.7	0.6	0.7	11, 11, 10
		333	10.0	2.0	0.6	8, 10, 12
		1000	12.7	6.0	0.8	19, 12, 7
		2500	11.3	1.5	0.7	10, 13, 11
		5000	6.7	1.5	0.4	5, 7, 8
	MNNG	5.0	203.7	24.0	12.7	194, 231, 186
TA 100	Water	-	114.3	19.4	-	131, 93, 119
	Test Item	33	120.7	11.4	1.1	108, 124, 130
		100	118.7	1.5	1.0	119, 117, 120
		333	120.3	11.0	1.1	124, 108, 129
		1000	121.0	6.6	1.1	122, 127, 114
		2500	113.7	16.8	1.0	110, 99, 132
		5000	112.3	12.9	1.0	98, 123, 116
	MNNG	5.0	1848.3	221.3	16.2	2102, 1695, 1748
TA 1537	Water	-	12.0	3.6	-	11, 16, 9
	Test Item	33	13.3	4.2	1.1	18, 10, 12
		100	13.7	5.5	1.1	14, 19, 8
		333	12.3	2.1	1.0	14, 10, 13
		1000	11.7	0.6	1.0	12, 11, 12
		2500	12.7	3.1	1.1	12, 10, 16
		5000	11.7	3.2	1.0	8, 14, 13
	AAC	100	141.0	19.1	11.8	129, 131, 163
TA 98	Water	-	25.7	5.7	-	32, 21, 24
	Test Item	33	17.0	4.0	0.7	13, 17, 21
		100	26.3	2.1	1.0	28, 24, 27
		333	27.3	7.6	1.1	19, 29, 34
		1000	30.3	4.0	1.2	28, 35, 28
		2500	19.7	4.6	0.8	17, 17, 25
		5000	20.3	8.7	0.8	13, 18, 30
	NOPD	10	1683.7	84.9	65.6	1718, 1587, 1746
E. coli	Water	-	26.7	6.8	-	19, 29, 32
	Test Item	33	29.0	4.0	1.1	25, 29, 33
		100	29.3	2.1	1.1	31, 27, 30
		333	30.7	5.9	1.2	24, 33, 35
		1000	28.0	3.6	1.1	29, 24, 31
		2500	24.7	4.5	0.9	29, 20, 25
		5000	15.3	6.7	0.6	23, 11, 12
	4-NQO	5	171.7	48.4	6.4	117, 189, 209

 

Table 5: Historical Negative Controls - SPT

Strain	S9 mix	Vehicle	No. of plates	No. of values	Min	Max	Mean	SD
TA 1535	without	all	324	131	6	23	14	3.0
	with	all	330	131	4	19	13	2.6
TA 100	without	all	342	132	82	202	117	15.4
	with	all	363	132	81	201	116	15.7
TA 1537	without	all	336	132	6	14	10	1.9
	with	all	339	132	6	16	10	1.9
TA 98	without	all	324	132	12	28	20	3.3
	with	all	348	132	16	33	24	3.4
E. coli	without	all	309	132	14	47	31	5.4
	with	all	309	132	18	50	32	7.5

 

Table 6: Historical Positive Controls - SPT

Strain	S9 mix	Pos. con.	No. of plates	No. of values	Min	Max	Mean	SD
TA 1535	without	MNNG	254	96	2152	7963	4519	1068.5
	with	2-AA	255	96	76	470	217	66.8
TA 100	without	MNNG	264	95	593	5718	3470	931.8
	with	2-AA	264	95	348	4066	2156	725.8
TA 1537	without	AAc	267	96	283	1653	1049	271.8
	with	1-AA	264	96	49	361	169	61.6
TA 98	without	NOPD	258	96	332	1060	516	111.3
	with	2-AA	261	95	350	3004	1580	564.0
E. coli	without	4-NQO	240	96	517	1902	1076	233.6
	with	2-AA	237	96	96	233	158	32.7

 

Table 7: Historical Negative Controls - PIT

Strain	S9 mix	Vehicle	No. of plates	No. of values	Min	Max	Mean	SD
TA 1535	without	all	276	102	8	24	13	2.7
	with	all	273	102	7	29	13	3.1
TA 100	without	all	281	103	75	150	114	14.9
	with	all	282	103	75	143	110	13.6
TA 1537	without	all	282	102	5	14	10	2.0
	with	all	279	102	5	17	10	2.0
TA 98	without	all	294	102	13	42	21	3.6
	with	all	282	102	17	35	25	3.5
E. coli	without	all	273	102	19	46	31	5.7
	with	all	273	102	17	45	30	5.9

 

Table 8: Historical Positive Controls - PIT

Strain	S9 mix	Pos. con.	No. of plates	No. of values	Min	Max	Mean	SD
TA 1535	without	MNNG	219	81	6651	5758	3273	800.3
	with	2-AA	2179	81	58	329	168	63.8
TA 100	without	MNNG	228	81	546	4583	2502	845.5
	with	2-AA	225	81	263	4056	1458	893.5
TA 1537	without	AAc	225	81	195	2240	944	315.6
	with	1-AA	225	81	45	419	141	61.5
TA 98	without	NOPD	234	81	376	797	526	88.2
	with	2-AA	225	81	163	3105	1138	648.1
E. coli	without	4-NQO	219	81	235	1574	634	364.0
	with	2-AA	219	81	99	276	167	38.1

Applicant's summary and conclusion

Conclusions:

negative

Executive summary:

In the GLP OECD471 study, hydroxypivalic acid neopentylglycol ester (HPN) was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

 

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5000 μg/plate (SPT); 33 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without external metabolic activation (liver S9 mix from rats treated with enzyme inducers).

SOLUBILITY: No precipitation of the test substance was observed with and without S9 mix.

TOXICITY: A bacteriotoxic effect was occasionally observed depending on the strain and test conditions at and above 2500 μg/plate

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (increased by a factor of 2 or above compared to the concurrent control for Salmonella typhimurium TA 100, TA 98 and Escherichia coli WP2 uvrA, or a factor of 3 or above for Salmonella typhimurium TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test with or without the addition of an external metabolizing system (liver S9 mix of rats treated with enzyme inducers).

 

Under the experimental conditions of this study, the test substance Hydroxypivalic acid neopentylglycol ester is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of external metabolic activation.