amporphous glass fibre formed from the melting and fiberisation of predominately slilcon dioxide, calcium oxide, magnesium oxide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed to recognised protocol by a GLP accredited laboratory

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

amporphous glass fibre formed from the melting and fiberisation of predominately slilcon dioxide, calcium oxide, magnesium oxide

EC Number:

610-130-5

Cas Number:

436083-99-7

Molecular formula:

Amorphous glass consisting of SinO(3n-1)2(n-1) Polymeric anions ionically bonded to Ca2+ and Mg2+ cations or other alkaline earth cations

IUPAC Name:

amporphous glass fibre formed from the melting and fiberisation of predominately slilcon dioxide, calcium oxide, magnesium oxide

Test material form:

solid: fibres

Details on test material:

- Name of test material (as cited in study report): AES Covance
- Physical state: white wool like solid
- Analytical purity: 100%
- Lot/batch No.: BG-10-X99-2983
- Expiration date of the lot/batch: 30 December 2020
- Stability under test conditions: stable
- Storage condition of test material: room temperature in sealed container

Method

Target gene:

hprt locus in L5178Y mouse lymphoma cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 media with different percentages of horse serum

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

(all in ug/ml)
Experiment 1
156.3
312.5
625
1250
2500
5000

Experiment 2
187.5
375
750
1500
3000
5000

Experiment 3
156.3
312.5
625
1250
2500
5000


Experiment 4
187.5
375
750
1500
3000
5000

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1% High viscocity Methyl Cellulose
- Justification for choice of solvent/vehicle: The Sponsor provided information to indicate that the test material AES Covance was an Alkaline Earth Silicate and it was therefore reliably assumed to be insoluble in all commonly used vehicles that are compatible with the assay system.

Controls

Untreated negative controls:

yes

Remarks:

cultures treated with culture medium alone

Negative solvent / vehicle controls:

yes

Remarks:

vehicle 1% MC diluted 10 fold in the treatment medium

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Remarks:

4-nitroquinoline-N-oxide positive control for expierments 1 & 2, benzo(a)pyrene positive control for experiments 3 & 4

Details on test system and experimental conditions:

METHOD OF APPLICATION: As suspension of test material in vehicle applied to cells in culture medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2x10^4 to 1x10^7

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative survival

OTHER EXAMINATIONS:
percentage relative survival; mutant frequency; viability; 6TG resistance

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in L5178Y mouse lymphoma cells if:
1. The MF at one or more concentrations was significantly greater than that of the vehicle control (p0.05)
2. There was a significant concentration relationship as indicated by the linear trend analysis (p0.05)
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

All calculations were performed by computer using validated software.
From the zero term of the Poisson distribution the probable number of clones/well (P) on microtitre plates in which there are EW empty wells (without clones) out of a total of TW wells is given by:
P = -ln (EW/TW).
Cloning Efficiency (CE) in any given culture is therefore:
CE = P/No of cells plated per well

Percentage Relative Survival (%RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus:
%RS = [CE (test)/CE (control)] x 100
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted as follows:
Adjusted %RS = %RS x Post-treatment cell concentration for test article treatment
Post-treatment cell concentration for vehicle control
All % RS values were adjusted as described above.
Mutant frequency (MF) is usually expressed as "mutants per 106 viable cells". In order to calculate this, the cloning efficiencies of both mutant and viable cells in the same culture were calculated:
MF = [CE (mutant)/CE (viable)] x 106
From the formulae given and with the knowledge that 2 x 104 cells were plated/well for mutation to 6TG resistance,
CE (mutant) = P (mutant)/2 x 104
CE (viable) = P (viable)/1.6
where, in each case, P = -ln (EW/TW).
Therefore,
MF = [P (mutant)/2 x 104] x [1.6/P (viable)] x 106
= {-ln [EW/TW (mutant)]/-ln [EW/TW (viable)]} x 80.
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A summary of the results for Experiments 1, 2, 3 & 4 is shown in the tables below. The acceptance criteria were met and the study was accepted as valid.
In Experiments 1 and 2, no statistically significant increases in MF were observed following treatment with AES Covance at any concentration tested in the absence of S 9 and there were no significant linear trends.
In Experiments 3 and 4, no statistically significant increases in MF were observed following treatment with AES Covance at any concentration tested in the presence of S 9 and there were no significant linear trends.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence of S‑9 ranging from 156.3 to 5000 mg/mL (an acceptable maximum concentration for in vitro genetic toxicology studies according to current regulatory guidelines). Upon addition of AES Covanceto the cultures, it was not possible to accurately determine the presence/absence of precipitate, as the test article was treated in suspension. Following the 3 hour treatment incubation period,precipitate and/or undissolved test article was observed at all concentrations tested. Due to the nature of the test article formulations, all cultures were retained. The highest concentration tested, 5000 µg/mL, gave 88% RS. The %RS values are shown in the following table.

No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations tested (5000mg/mL) as compared to the concurrent vehicle controls (individual data not reported).

Concentration	%RS
µg/mL	-S-9
0	100
UTC	152
156.3	125
312.5	153
625	100
1250	109
2500	100
5000	88

UTC       Untreated control

%RS       %Relatvive survival adjusted by post treatment cell counts

No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations tested (5000mg/mL), compared to the concurrent vehicle controls (individual data not reported).

Experiment 1

In Experiment 1 six concentrations, ranging from 156.3 to 5000 µg/mL, were tested in the absence of S‑9. Upon addition of AES Covance to the cultures, it was not possible to accurately determine the presence/absence of precipitate, as the test article was treated in suspension. Following the 3 hour treatment incubation period, precipitate and/or undissolved test article was observed at all concentrations tested. Due to the nature of the test article formulations, all cultures were retained. Seven days after treatment all concentrations were selected to determine viability and 6TG resistance. The highest concentration analysed, 5000 µg/mL, gave 66% RS (see following table).

Concentration	%RS	MF §
µg/mL
0	100	2.48
UTC	99	1.66
156.3	101	2.06 NS
312.5	93	1.85 NS
625	93	1.59 NS
1250	94	1.13 NS
2500	65	1.36 NS
5000	66	2.83 NS
NQO 0.15	48	27.52
NQO 0.20	34	70.82

Linear Trend test: Not significant

 

UTC       Untreated control

§             6‑TG resistant mutants/10⁶viable cells 7 days after treatment

%RS       Percent relative survival adjusted by post treatment cell counts

NS          Not significant

 

Experiment 2

In Experiment 2 six concentrations, ranging from 187.5 to 5000 µg/mL, were tested in the absence of S‑9. Upon addition of AES Covance to the cultures, it was not possible to accurately determine the presence/absence of precipitate, as the test article was treated in suspension. Following the 3 hour treatment incubation period, precipitate and/or undissolved test article was observed at all concentrations tested. Due to the nature of the test article formulations, all cultures were retained. Seven days after treatment all concentrations were selected to determine viability and 6TG resistance. The highest concentration analysed, 5000 µg/mL, gave 54% RS (see following table).

 

Concentration	%RS	MF §
µg/mL
0	100	4.71
UTC	81	3.26
187.5	100	1.34 NS
375	96	2.75 NS
750	79	2.87 NS
1500	71	3.36 NS
3000	77	2.83 NS
5000	54	1.57 NS
NQO 0.15	60	48.02
NQO 0.20	26	75.65

Linear Trend test: Not significant

 

UTC       Untreated control

§             6‑TG resistant mutants/10⁶viable cells 7 days after treatment

%RS       Percent relative survival adjusted by post treatment cell counts

NS          Not significant

Experiment 3

In Experiment 3 six concentrations, ranging from 156.3 to 5000 µg/mL, were tested in the presence of S‑9. Upon addition of AES Covance to the cultures, it was not possible to accurately determine the presence/absence of precipitate, as the test article was treated in suspension. Following the 3 hour treatment incubation period, precipitate and/or undissolved test article was observed at all concentrations tested. Due to the nature of the test article formulations, all cultures were retained. Seven days after treatment all concentrations were selected to determine viability and 6TG resistance. The highest concentration analysed, 5000 µg/mL, gave 67% RS (see following table).

3 hour treatment in the presence of S-9

Concentration	%RS	MF §
µg/mL
0 PP	100	4.06
UTC	121	3.46
156.3 PP	87	3.82
312.5 PP	110	3.94
625 PP	75	2.91
1250 PP	61	1.47
2500 PP	61	3.27
5000 PP	67	6.17
B[a]P 2 PP	71	15.81
B[a]P 3 PP	46	29.58

 

Test for Linear Trend

Slope	3.05E-10
Variance	6.95E-20
b² / Sb	1.336

Experiment 4

In Experiment 4 six concentrations, ranging from 187.5 to 5000 µg/mL, were tested in the presence of S‑9. Upon addition of AES Covance to the cultures, it was not possible to accurately determine the presence/absence of precipitate, as the test article was treated in suspension. Following the 3 hour treatment incubation period, precipitate and/or undissolved test article was observed at all concentrations tested. Due to the nature of the test article formulations, all cultures were retained. Seven days after treatment all concentrations were selected to determine viability and 6TG resistance. The highest concentration analysed, 5000 µg/mL, gave 63% RS (see following table).

3 hour treatment in the presence of S-9

Concentration	%RS	MF §
µg/mL
0	100	3.58
UTC	86	2.54
187.5 PP	103	5.73
375 PP	101	2.59
750 PP	87	6.26
1500 PP	81	6.33
3000 PP	67	3.42
5000 PP	63	5.19
B[a]P 2	94	17.96
B[a]P 3	59	29.54

 

Test for Linear Trend

Slope	1.77E-10
Variance	6.16E-20
b² / Sb	0.509

 

UTC                       Untreated control

§                             6‑TG resistant mutants/10⁶viable cells 7 days after treatment

%RS                       Percent relative survival adjusted by post treatment cell counts

PP                          Precipitation noted at end of treatment incubation period

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that AES Covance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to 5000 g/mL, an acceptable maximum concentration for this type of study according to current regulatory guidelines, both in two independent experiments in the absence of metabolic activation and two independent experiments in the presence of metabolic activation..

Executive summary:

AES Covance was assayed for the ability to induce mutation at the hypoxanthine‑guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by four independent experiments, two conducted in the absence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S‑9) and two conducted in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S‑9).

All AES Covance treatments in this study were performed using formulations prepared as a suspension in 1% high viscosity methyl cellulose (1% MC). As the treatment formulations were in suspension, all concentrations cited in this report may be regarded as nominal.

A 3 hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence of S‑9, ranging from 156.3 to 5000 µg/mL (an acceptable maximum concentration for in vitro genetic toxicology studies according to current regulatory guidelines). The highest concentration tested, 5000 mg/mL, gave 88% RS.

In Experiment 1 six concentrations, ranging from 156.3 to 5000 µg/mL, were tested in the absence of S‑9. Seven days after treatment the highest concentration analysed to determine viability and 6TG resistance, 5000 µg/mL, gave 66% RS.

In Experiment 2 six concentrations, ranging from 187.5 to 5000 µg/mL, were tested in the absence of S‑9. Seven days after treatment the highest concentration analysed to determine viability and 6TG resistance, 5000 µg/mL, gave 54% RS.

In Experiment 3 six concentrations, ranging from 156.3 to 5000 µg/mL, were tested in the presence of S‑9. Seven days after treatment the highest concentration analysed to determine viability and 6TG resistance, 5000 µg/mL, gave 67% RS.

In Experiment 4 six concentrations, ranging from 187.5 to 5000 µg/mL, were tested in the presence of S‑9. Seven days after treatment the highest concentration analysed to determine viability and 6TG resistance, 5000 µg/mL, gave 63% RS

Vehicle and positive control treatments were included in each Mutation Experiment. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4‑nitroquinoline 1-oxide (NQO) and benzo(a)pyrene. Therefore the study was accepted as valid.

In Experiments 1 and 2, no statistically significant increases in MF were observed following treatment with AES Covance at any concentration tested in the absence of S‑9 and there were no significant linear trends.

In Experiments 3 and 4, no statistically significant increases in MF were observed following treatment with AES Covance at any concentration tested in the presence of S‑9 and there were no significant linear trends.

It is concluded that AES Covance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to 5000mg/mL, an acceptable maximum concentration for this type of study according to current regulatory guidelines, in four independent experiments both in the absence and presence of metabolic activation.