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Three studies were conducted with the read across substance boron trifluoride. For this, boron trifluoride dihydrate, was tested in an Ames test (May K., 2010), a mouse lymphoma assay (Woods I., 2010a) and an in vitro aberration chromosomal test (Woods I., 2010b). Additionally, two studies with phosphoric acid (Ames test and a mouse lymphoma assay) were used for read across.

 

Ames test (boron trifluoride dihydrate)

In this in vitro assessment of the mutagenic potential of boron trifluoride dihydrate (according to OECD 473, GLP) histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101) were exposed to boron trifluoride dihydrate diluted in water. Water was also used as a negative control. Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of boron trifluoride dihydrate up to 5000 µg/plate were tested. No signs of toxicity were observed towards the tester strains in the first mutation test. Toxicity (observed as slight thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers) was seen in all strains following exposure to boron trifluoride dihydrate at 5000 µg/plate in the second mutation test. No evidence of mutagenic activity was seen at any concentration of boron trifluoride dihydrate in either of the two experiments. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory. In conclusion, boron trifluoride dihydrate showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

 

Ames test (phosphoric acid)

In this reverse gene mutation assay in bacteria according to OECD guideline 471, strains of S. typhimurium (TA 98, TA100, TA1535, TA1537) and E. coli (WP2uvrA) were exposed to the read across substance phosphoric acid at concentrations of 50 to 5000 µg/plate with and without metabolic activation (S9 mix). Two independent experiments were performed using the plate incorporation (Experiment 1) and the pre-incubation assay (Experiment 2). Each concentration as well as the vehicle and positive controls were tested in triplicate.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

The test material was considered to be non-mutagenic under the conditions of this test.

 

Mouse lymphoma assay (boron trifluoride dihydrate)

Boron trifluoride dihydrate was tested for mutagenic potential in an in vitro mammalian cell mutation assay (OECD 476, GLP). The study consisted of a preliminary toxicity test and two main tests comprising three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. Boron trifluoride dihydrate was found to be soluble at 67.8 mg/mL in water. A final concentration of 450 mg/mL, dosed at 1%v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the highest concentration that does not cause a fluctuation in pH of more than 1.0 unit. Toxicity was observed in the preliminary toxicity test.

Following a 3-hour exposure to boron trifluoride dihydrate at concentrations from 0.9 to 450 mg/mL, relative suspension growth (RSG) was reduced from 107 to 71% and from 113 to 68% in the absence and presence of S9 mix, respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 114 to 1%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG), or to assess exposure up to the highest concentration that does not cause a fluctuation in pH of more than 1.0 unit. Following 3-hour treatment in the absence and presence of S9 mix, there were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix was 450 mg/mL. In the absence and presence of S9 mix RTG was reduced to 51 and 46%, respectively. In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 300 mg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF was observed at concentrations up to 300 mg/mL, where RTG was reduced to 11%. In all tests the concurrent vehicle and positive control were within acceptable ranges. It was concluded that boron trifluoride dihydrate did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

 

Mouse lymphoma assay (phosphoric acid)

Phosphoric acid was tested for mutagenic toxicity in an in vitro mammalian cell mutation assay according to OECD guideline 476. The study consisted of a preliminary test and two main tests comprising three independent mutagenicity assays. The cells were exposed for either 4 hours with and without metabolic activation or for 24 hours without metabolic activation.

In the three exposure groups (4-h exposure -S9, 4-hour exposure +S9 and 24-hour exposure -S9) of the preliminary test there were no marked dose related reductions in the Relative Suspension Growth (%RSG) of treated cells with the test material when compared to the concurrent vehicle controls. However, a modest reduction was observed at the 10 mM limit dose in all three of the exposure groups with the greatest reduction observed in the 24-hour exposure group in the absence of metabolic activation.

The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG), or to assess exposure up to the highest concentration that does not cause a fluctuation in pH of more than 1.0 unit.

In the main test the cells were exposed to the test substance at concentrations of 0, 61.25, 122.5, 245, 490, 735 and 980 µg/mL.

Following 4-hour treatment in the absence and presence of S9 mix, there were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency.

The maximum concentrations assessed for mutant frequency in the 4 hour treatment in the absence and presence of S9 mix was 980 µg/mL. In the absence and presence of S9 mix RTG was reduced to 74 and 81 %, respectively.

In the 24 hour treatment, the maximum concentration assessed for mutant frequency was also 980 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency was observed at concentrations up to 980 µg/mL, where RTG was reduced to 53%. In all tests the concurrent vehicle and positive control were within acceptable ranges. It was concluded that the test substance did not demonstrate mutagenic potential in this in vitro cell mutation assay under the experimental conditions described.

 

Chromosome Aberration assay (boron trifluoride dihydrate)

A study was performed to assess the ability of the read across substance boron trifluoride dihydrate to induce chromosomal aberrations in human lymphocytes cultured in vitro (OECD 473, GLP).

Human lymphocytes were exposed to the test substance both in the absence and presence of S9 mix. Solvent and positive control cultures were also included. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

In order to determine the toxicity of the boron trifluoride dihydrate to cultured human lymphocytes, the mitotic index was assessed for all cultures treated with the test substance and the solvent control (water). A final concentration of 450 mg/mL, dosed at 1% v/v, was used as the maximum concentration, in order to test up to the highest concentration that does not cause a fluctuation in pH of more than 1.0 unit. On the basis of this data, the following concentrations were selected for metaphase analysis:

First test

In the absence of S9 mix: 3-hour treatment, 18-hour recovery: 162, 270 and 450 µg/mL

In the presence of S9 mix (2% v/v): 3-hour treatment, 18-hour recovery: 162, 270 and 450 µg/mL

Second test

In the absence of S9 mix: 21-hour continuous treatment: 97.2, 162 and 270 µg/mL.

In the presence of S9 mix (5% v/v): 3-hour treatment, 21-hour recovery: 162, 270 and 450 µg/mL

 

In the absence of S9 mix, boron trifluoride dihydrate caused statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at 450 µg/mL (3-hour treatment, including gaps; no significant increase when excluding gaps) and at 162 µg/mL (21-hour treatment, including gaps; no significant increase when excluding gaps) and 270 µg/mL (21-hour treatment, excluding and including gaps), when compared with the concurrent solvent control.

In the presence of S9 mix, boron trifluoride dihydrate caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control, in either test.

No statistically significant increases in the proportion of polyploid cells were observed during metaphase analysis in either test.

All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

In conclusion boron trifluoride dihydrate has shown evidence of causing an increase in the frequency of structural chromosome aberrations in the absence of S9 mix after 21-hour exposure period under the experimental conditions described.

 

Conclusions

Both Ames test and mouse lymphoma assay with the two read across substances did not show any mutagenic activity up to the highest tested concentrations.

But the in vitro chromosomal aberration test with boron trifluoride dihydrate gave inconclusive results. In the absence of S9 mix, boron trifluoride dihydrate caused statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at 270 µg/mL (21-hour treatment, excluding and including gaps), when compared with the concurrent solvent control.

In the presence of S9 mix, boron trifluoride dihydrate caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control, in either test.

In conclusion, Boron Trifluoride Dihydrate has shown evidence of causing an increase in the frequency of structural chromosome aberrations, in the absence of S9 mix, including and excluding gaps, but only after 21-hour exposure period. This increase is accompanied by a low mitotic index of 51 %.

Furthermore the mouse lymphoma assay is able to detect certain clastogenic effects, but none were observed in the current test. The in vitro chromosomal aberration test should be regarded as ambiguous.

Follow-up action regarding the ambiguous chromosomal aberration test with boron trifluoride dihydrate:

To clarify the ambiguous results of the chromosomal aberration test with boron trifluoride dihydrate, an in vivo micronucleus test would be necessary. The feasibility of such a test was discussed between the registrants of boron trifluoride (CAS 7637-07-2) and the European Chemicals Agency (ECHA) in the course of a dossier compliance check for boron trifluoride (registration number 01-2119534579-27-0000).

On July 5th, 2012 ECHA has taken the decision (based on a proposal from the Registrants) that an in vivo micronucleus test via inhalation route should be performed with boron trifluoride dihydrate by the registrant in accordance with the procedure set out in Articles 50 and 51 of Regulation (EC) No. 1907/2006. The investigation has been started with a technical trial, however, the pilot experiments and recalculations (reported below) let the Registrants ask ECHA for a reconsideration of the decision for animal welfare reasons. Indeed, it has been shown that even at low atmospheric concentrations the substance will be an aerosol liquid whose corrosive properties would generate severe focal damage. In addition, it will not be possible to achieve the concentration leading to the increase in the frequency of structural chromosome aberrations observed in vitro (See genetic toxicity in vivo-waiver).

The registrant argued that the current German occupational exposure limits (OELs) are set at 1 mg/m3 for boron trifluoride (being 600 fold below the needed concentration to receive an effective dose in the blood). The high concentrations that need to be tested within the in vivo MNT will according to the registrant's calculations not lead to observable effects and will not decrease the DNEL below these OELs. For the reasons explained above, no in vivo mutagenicity tests including the in vivo MNT as well as other in vivo tests such as the Comet Assay should be conducted with BF3.

By letter on the 28th January 2014, Arkema as Lead Registrant was informed that a Statement Of Non Compliance (SONC) was issued against them. Indeed, pursuant to Article 41(3) of Rgulation (EC) 1907/2006 (REACH Regulation) the European Chemicals Agency (ECHA) has performed a testing proposal examination on the dossier boron trifluoride, CAS No 7637 -07 -2, EC No. 231 -569 -5 and taken the decision TPE-D-0000002344 -80 -04/F. In that decision, the Registrant was requested to submit by 5th July 2013 information on the following issue: Mammalian erythrocyte micronucleus test, inhalation route, (Annex IX, 8.4., test method : EU B.12/OECD 474) according to certain conditions.

ECHA has examined the information submitted in the update dossier on the 27th June 2013 and considered that the updated registration dossier did not contain the information requested by the ECHA decision as a waiving justification was provided instead of experimental data. ECHA did not consider the rationale for this waiving justification was provided instead of experimental data. ECHA did not consider the rationale for this waiving as being sufficient;

Please find below the registrants’ answers regarding the following ECHA comments:

-" First, the Registrant refers to pilot experiments and calculations performed which he states are attached. However, the attachements could not be found in the technical dossier. Therefore, it is not clear on what arguments the registrant has based his adaptation".

Answer of the Registrant:

Please find attached the report of this experiment (12I048 -SR-LM-4) which demonstrated that the exposure atmosphere could have been almost exclusively composed of BF3 -2H2O aerosol. Under the current study conditions, a high aerosol fraction was detected at a dosing rate as low as 1.8 mL/h. In the range of LC50 value, BF3 is present as liquid aerosol in atmosphere. In addition, the sampling method for gas and vapours failed to provide reliable data for the atmosphere concentration due to a high absorption onto the chamber walls. Since the substance is corrosive, and given that the test atmosphere will be almost exclusively composed of BF3 -2H2O aerosol, it is evident that local corrosive effects would be induced at each impaction area as the substance would be available almost undiluted at each contact site.

By considering the animal welfare, the Study Director has taken the decision not to perform the study (see Boron trifluoride statement document) which is in line with the REACH and OECD guidelines. Specifically, Article 13(4) of Reach requires that toxicological and ecotoxicological tests shall be carried out in compliance with EU Directive 2010 -63 -EU on animal protection which sets out the basic requirements for the care and accomodation of Laboratory animals, and stipulates that experiments shall be designed to avoid distress and unnecessary pain and suffering to the animal. In addition, according to the general part of Annexes VII-X, in vivo testing with corrosive substances at concentration/dose levels causing corrosivity should be avoided to the extent possible.

-" Secondly, the Registant states that it is not possible to obtain concentration in vivo that would be similar to the concentrations used in the in vitro study that caused an increase in structural chromosomal aberrations due to the corrosive nature of the substance. The OECD/EU test guideline specifies that test concentrations should be selected to "cover a range the maximum to little or no toxicity observed, and further"... the highest dose may also be specified as a dose that produces some indications of toxicity of bone marrow..." This means that toxicity of bonne marrow is not a pre-requisite for the dose selection. The dossier contains studies that have been performed in vivo via inhalation route, where toxicity was observed already at low doses e.g. 6 mg/m3 (repeated dose toxicity study, kidney effects). Respiratory distress was noted in all doses (2 to 180 mg/m3), however with no abnormal histological findings. Moreover, the direct comparison between concentrations used in in vitro studies and doses to be used in in vivo study cannot be made, due to the differences in test systems. Therefore, ECHA considers that the Registrant's reasoning according to which " high enough dose levels cannot be obtained" is not supported by evidence.

Answer of the Registrant:

In the final decision TPE-D-0000002344 -80 -04/F, ECHA emphasizes that the Registrant needs to demonstrate that the test method is applicable for the test substances taking into account specifically paragraph 7 of the OECD TG 474 ("if there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not apppropriate to use this test"). In view of the results of the experimental trial and the in vivo data indicating significant toxicity even at low doses, it became evident that the selection of dose-levels will be significantly low as well the low probability that the test substance reaches the bonne marrow. Thus, taking into account the animal welfare considérations and the pertinence of the test, a waiving statement was written by the Study Director.

Now, it appears that the comments made by ECHA in the SONC are in contradiction with the previous comments. ECHA seems now to consider that there is no need to prove the reaching of the target tissue (i.e. the bone marrow) and that the observation of clinical or systemic effects (kidney findings) which could indicate that the bone marrow has been reached were observed at high dose-levels in only few animals.

- Lastly, the Registrant refers to German OEL of 1 mg/m3 and the potential 600 time higher concentration (not clear if the Registrant's calculation is based on the in vitro study) that would need to be used to obtain an "effective dose in blood". The Registrant's conclusion is that the results obtained by using these high concentrations would not change the DNEL below the OEL. However, this statement has two drawbacks. First, hazard data from either animal studies or human evidence is needed before DNELs can be calculated. Therefore, before the data is available, it is impossible to know if DNELS or OELs would need to be changed. Moreover, structural chromosomal aberrations are considered to be following non threshold mechanisms; hence a dose without potential effects (DNEL) cannot be derived from such data. Consequently, the Registrant's arguments concerning the potential concentrations used and reference to DNELs is not accepted by ECHA.

Answer of the Registrant:

We agree with the fact that we cannot anticipate the results of a study, however, we firmly believe that whatever the result of the micronucleus test, this will not impact the systemic inhalation long term exposure DNEL. Firstly, all the available data indicate that the critical short- or long-term effect is the highly corrosive potential observed since low exposure and the existing DNEL is based on this effect. Secondly, in the absence of reliable chronic study data and/or elements that could suggest a carcinogen popential, no extrapolation to humans can be made.

Lastly, as the toxicity of the substance requires Risk Management Measures and low occupational exposure limits, there is no concern regarding the carcinogen potential of the substance.

 

In conclusion, a valid and guideline-compliant in vivo micronucleus test with the boron trifluoride phosphoric acid complex is not considered scientifically justified for the reasons described above.


Short description of key information:
Five in vitro studies are available for the read-across substances boron trifluoride and phosphoric acid. All were conducted according to OECD guidelines and are GLP. Ames test and mouse lymphoma assay are negative for both substances. The in vitro mammalian chromosome aberration test in human lymphocytes with boron trifluoride dihydrate is positive but only without S9 mix and for a 21-hour exposure period (including and excluding gaps).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance does not need to be classified and labelled for mutagenic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EC.

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled for mutagenic toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation EC No 605/2014.

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