Reaction Mass of 4-tert-butyl-2-hydroxycyclohexyl methacrylate and 5-tert-butyl-2-hydroxycyclohexyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES Test: Negative (with or without metabolic activation); OECD 471 (GLP); Riach (1999)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 1998 - 17 February 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine (G46, C3076 and D3052) - S. typhimuriumtryptophan+ reversion system - E. coli

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

With S9 mix - 5, 16.7, 50, 166.7, 500 and 1666.7 µg/plateWithout S9 mix - 1.7, 5, 16.7, 50, 166.7 and 500 µg/plateToxicity test (with and without S9 mix) - 16.7, 50, 166.7, 500, 1666.7 and 5000 µg/plate

Vehicle / solvent:

DMSO - 100 µL (with and without S9 mix)

Untreated negative controls:

yes

Remarks:

Each bacterial strain was tested for its resistance to ampicillin (indicating the presence of pkM101) and sensitivity to UV light and crystal violet (indicating persistence of the uvrB and rfa mutations)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

In the course of testing, 2 methods of treatmetn were performed to extend the range of the conditions of the assay. The tests were performed using the Direct Plate method and Pre-incubation method.Direct Plate method2 mL of soft agar was suspended in a small sterile tube. To this, 0.5 mL of S9 mix or 0.05 M phosphate buffer (pH 7.4) was added, followed by 0.1 mL of bacteria. The solvent or test solution was added last. The tubes were mixed and poured on to agar plates (25 mL 1.5 % purified agar in Vogel-Bonner E with 2 % glucose. The plates were inverted after the soft agar had set and incubated at 37 °C for 2 days.Pre-incubation method0.5 mL of S9 mix or 0.05M phosphate buffer (pH 7.4) was ispensed into a small sterile tube. This was followed by 0.1 mL of bacteria and finally the test solution or solvent (0.1 mL also). The tube was then incubated at 37 °C for 20 mins after which time 2 mL of soft agar was added. The contents were mixed then poured onto Vogel-Bonner plates. The plates were inverted and incubated for 2 days at 37 °C after the soft agar had set.In both methods the bacterial colonies were counted and plates examined for precipitates and microscopic colony growth.A toxicity test was conducted using TA 100 only and was performed in the presence and absence of S9 mix to establish suitable dose levels for the mutation test.The mutation tests were conducted with all bacterial strains in the presence and absence of S9 mix using the two methods outlined above. In the presence of S9 mix, the test was conducted at 5, 16.7, 50, 166.7, 500 and 1666.7 µg TBE/plate. In the absence of S9 mix, the test was conducted at 1.7, 5, 16.7 50, 166.7 and 500 µg TBE/plate. Triplicate plates were prepared at each concetration for each strain of bateria in the presence and absence of S9 mix.Positive control samples were prepared as follows;The activity of the S9 mix and mutability of the bacteria was demonstrated by plating E coli with 20 µg 2-AAN (2-aminoanthracene)/plate, TA 1535 and TA 1537 with 2 µg 2-AAN/plate and TA 98 and TA 100 with 0.5 µg 2-AAN/plate. This test was conducted in the presence of S9 mix.Additional plates were prepared in the absence of S9 mix and contained N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) in the presence of E. coli (2 µg ENNG/plate); sodium azide (NaN3) in the presence of TA 1535 and TA 100 (1 µg NaN3/plate); 2-nitrofluorene (2-NF) in the presence of TA 98 (1 µg 2-NF/plate); 9-aminoacridine (9-AA) in the presence of TA 1537 (80 µg 9-AA/plate). These plates served as an aid to strain identification and to demostrate the mutability of the bacteria.Negative controls were prepared which tested each strain of bacteria for its resistance to ampicillin (indicating the presence of pkM101) and sensitivity to UV light and crystal violet (indicating persistence of the uvrB and rfa mutations).

Evaluation criteria:

1. the bacteria demostrated their typical response to crystal violet, ampicillin and UV light.2. at least 2 of the vehicle control plates were within the follow the following ranges; TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and E. coli, 1-60.3. on at least 2 of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5 the mean vehicle control mutant number per plate. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required on at least 2 of the positive control plates.4. no toxicity or contamination was observed in at least 4 dose levels.5. in cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number.If the criteria above were met, a significant mutagenic response was recorded if there was;1. for TA 1535, TA 1537, TA 98 and E. coli, at least a doubling of the mean concurrent vehicle control values at some concentration of the test substance (1.5 x for TA 100). If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes.2. a dose related response3. a reproducible effect in independent tests

Statistics:

No statistics were used when interpreting the results.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TBE did not induce mutagenic activity in any of the 5 bacterial strains tested, in either the presence or absence of S9 mix.Precipitation of the test item was observed at 1666.7 µg /plate (this concetration was used in the presence of S9 mix).TBE was toxic to bacteria, generally from 500 µg/plate.Positive control groups were within the normal rnages expected for each bacterial strain and activation criteria.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

It was concluded that TBE was not mutagenic to S. typhimurium or E. coli when tested in DMSO at concetrations extending into the toxic range.

Executive summary:

TBE was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Eschericha coli WP2uvrA at concetrations ranging from 1.7 to 1666.7 µg/plate.

Tests were conducted on agar plates using both the Direct Plate and Pre-incubation method in the presence and absence of S9 mix.

Concurrent positive controls demostrated the sensitivity of the assay and the metabolising activity of the S9 mix.

No mutagenic response was observed in any of the 5 bacteria strains, in either the presence or absence of S9 mix.

Precipitation of tTBE was observed at 1666.7 µg/plate. TBE was also found to be toxic to bacteria, generally, around 500 µg/plate.

It was concluded that TBE was not mutagenic to S. typhimurium or E. coli when tested in DMSO (dimethylsulphoxide) at concetrations extending into the toxic range.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with CLP (1272/2008, as amended) criteria, the substance does not meet the criteria for mutagenicity based on available data. Available data are consistent with REACH information requirements.