Occupational Exposure to Methylene Chloride

DEPARTMENT OF LABOR

Occupational Safety and Health Administration

29 CFR Parts 1910, 1915 and 1926

[Docket No. H-071B]

Occupational Exposure to Methylene Chloride

AGENCY: Occupational Safety and Health Administration (OSHA),

Department of Labor.

ACTION: Proposed rule; limited reopening of the rulemaking record.

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SUMMARY: The Occupational Safety and Health Administration (OSHA) is

reopening the record for the proposed revision of the methylene

chloride (MC) standard (56 FR 57036, November 7, 1991) for comments on

recently conducted research regarding MC metabolism. OSHA's proposed MC

risk assessment for cancer was based primarily on extrapolations from

mouse bioassay data. The proposal and the hearing notice (57 FR 24438,

June 9, 1992) solicited input regarding the relevance of metabolic and

physiological differences between mice and humans when assessing human

cancer risk. As a result, the rulemaking record already contains

considerable information, comment and testimony regarding this issue.

The new studies address the potential pathway(s) by which MC

metabolites induce lung and liver cancer in mice and draw conclusions

regarding the relevance of the mouse data to the assessment of human

cancer risk. OSHA has determined that these studies are relevant to

full consideration of concerns raised by the MC rulemaking. Therefore,

OSHA is reopening the record to allow the public an opportunity to

comment.

DATES: Written comments on the materials incorporated through the

notice of reopening must be postmarked by November 24, 1995.

ADDRESSES: Comments are to be submitted in quadruplicate to the Docket

Office, Docket No. H-071B, U.S. Department of Labor, Room N-2634, 200

Constitution Avenue, NW, Washington, DC 20210. telephone (202) 219-

7894. Written comments limited to 10 pages or less in length may also

be

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transmitted by facsimile to (202) 219-5046, provided that the original

and 3 copies are sent to the Docket Office thereafter.

FOR FURTHER INFORMATION CONTACT: Anne C. Cyr, Office of Information and

Consumer Affairs, Occupational Safety and Health Administration, U.S.

Department of Labor, Room N-3647, 200 Constitution Avenue, NW,

Washington, DC 20210. Telephone (202) 219-8148. Copies of the

referenced studies ae available for inspection and copying in the

Docket Office and will be immediately mailed to persons who request

copies by telephoning Christine Whittaker at (202)219-7174. For

electronic copies, contact the Labor News Bulletin Board (202) 219-

4784; or OSHAs WebPage on Internet at http://www.osha.gov/. For news

releases, fact sheet, and other short documents, contact OSHA FAX at

(900) 555-3400 at $1.50 per minute.

SUPPLEMENTARY INFORMATION:

I. Background

On November 7, 1991, OSHA issued a notice of proposed rulemaking

(56 FR 57036) to address the significant risks of MC- induced health

effects. The proposed rule required employers to reduce occupational

exposure to MC and to institute ancillary measures, such as employee

training and medical surveillance, for further protection of MC-exposed

workers.

OSHA convened public hearings (57 FR 24438, June 9, 1992) in

Washington, DC on September 16-24, 1992 and in San Francisco, CA on

October 14-16, 1992. The post-hearing period for the submission of

additional briefs, arguments and summations ended on March 15, 1993. On

March 11, 1994, OSHA reopened the rulemaking record for 45 days (59 FR

11567) to obtain public input on three documents incorporated into the

rulemaking record, one of which examined the relationship between MC

exposure and human carcinogenesis. The limited reopening, which ended

on April 25, 1994, generated 37 comments.

OSHA relied primarily on the mouse bioassay performed by the

National Toxicology Program (NTP) in assessing human cancer risks in

the proposed rule. The Preliminary Quantitative Risk Assessment was

based on a multistage model which used the applied dose from the NTP

study in the dose-response analysis. The proposal and the hearing

notice solicited input regarding the extent to which metabolic

differences between mice and humans could be taken into account when

assessing human cancer risk. The Agency generated a considerable amount

of information, comments and testimony regarding this issue at the

public hearings and in the post-hearing comment periods. Thus the

rulemaking record upon which the final risk assessment will be based

already includes substantial data for analysis using either

administered-dose or pharmacokinetic models.

In September 1995, the Halogenated Solvents Industry Alliance

(HSIA) submitted several recently-completed studies on this issue in

which HSIA asserted that species differences in the enzymatic

metabolism of MC make the mouse a poor surrogate for estimating human

cancer risk. The utility of the mouse data in assessing human risk is

one of the important issues in this rulemaking. Therefore, OSHA

believes that it is appropriate, even at this late stage of the

rulemaking process, to consider the HSIA-submitted studies in the

drafting of the final rule. Accordingly, the Agency is reopening the

rulemaking record to incorporate these studies and to provide the

public with an opportunity to comment.

As discussed above, OSHA has been considering the impact of species

differences on the MC risk assessment throughout this rulemaking, and

has generated an extensive record over the nearly four years since the

proposal was published. While the Agency agrees with HSIA that the new

materials should be taken into account, the Agency still believes that

every reasonable effort should be made to finish this rulemaking

expeditiously. To that end, OSHA has concluded that it is appropriate

to allow interested parties 30 days within which to submit any

additional comments and information regarding this issue. OSHA will

provide interested parties with copies of the newly incorporated

materials, upon request, to facilitate full and timely public

participation. Requests for copies should be addressed to Christine

Whittaker, Room N-3718, Health Standards Programs, OSHA, U.S.

Department of Labor, 200 Constitution Avenue, NW, Washington, DC 20210.

Telephone: (202) 219-7174. Fax: (202) 219-7125.

In addition, OSHA notes that HSIA has submitted data on lung tissue

obtained through the Zeneca Toxicology Laboratory in a preliminary

communication (Ex. 124) but has not yet submitted a final report of

this research. The Agency has determined, given the availability of the

preliminary communication, that it would be inappropriate to delay the

reopening until the final report was received. HSIA has indicated that

the report will be submitted during the reopening period. As with the

materials already docketed, OSHA will provide copies of that report,

upon request, when it becomes available.

The materials added to the record consist of a transmittal letter

from HSIA and seven technical submissions as discussed below.

Exhibit 117 Letter from Peter E. Voytek, Ph.D., of the Halogenated

Solvents Industry Alliance to Joseph A. Dear, Assistant Secretary of

Labor for Occupational Safety and Health, September 5, 1995

This letter introduces the HSIA studies covered by the notice of

reopening and requests that OSHA ``reopen the rulemaking record for the

limited purpose of obtaining public comment (and additional scientific

peer review, to the extent OSHA deems it appropriate) on this

evidence.'' In particular, the HSIA letter states that the mice used in

studies on which OSHA's risk assessment is based ``are uniquely

sensitive at high exposure levels to methylene chloride-induced lung

and liver cancer, and that other species, including humans, are not at

similar risk.'' The letter summarized the basis for this interpretation

as follows:

As a result of this research program, it appears that there are

no foreseeable conditions of human exposure in which the

carcinogenic effects seen in mice could be expected to occur in man.

Given the unique metabolism of methylene chloride by mice, the mouse

cannot be considered an appropriate model for human risk assessment.

The risk assessment that is the basis for the methylene chloride

standard, which is in turn based on the increased liver and lung

tumor incidence observed in the mouse bioassay, must be discarded in

favor of scientific data that are relevant to human risk.

OSHA requests that commenters review the following technical

studies to assess whether the conclusions summarized above are

appropriate, in light of the evidence contained therein, considering

factors such as: (1) The relevance, reliability, and sensitivity of the

assays used (e.g., the DNA single-strand break assay reported in

Exhibit 120 and the mRNA assay reported in Exhibit 124); (2) the

existing evidence in the record indicating quantitative differences in

MC metabolism between mice and humans; (3) the weight of evidence

contributed by these in vitro studies evaluated in light of the other

in vitro and in vivo information already in the record; and (4) other

relevant factors. The Agency also requests that commenters address the

extent to which these studies might also support alternative

conclusions.

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Exhibit 118 ``Methylene Chloride Induced Mouse Liver and Lung

Tumours'', T. Green, Zeneca Central Toxicology Laboratory, July 31,

1995

This document summarizes the available information regarding the

metabolism of methylene chloride in mice, rats, hamsters and humans.

The researcher characterized this information as follows: ``These

results provide evidence that the mouse is unique in its response to

methylene chloride and that it cannot therefore be considered an

appropriate model for human health assessment.''

Exhibit 119 ``Methylene Chloride: an inhalation study to investigate

toxicity in the mouse lung using morphological, biochemical and Clara

cell culture techniques,'' J.R. Foster, T. Green, L.L. Smith, S.

Tittensor, and I. Wyatt, Toxicology 91 (1994) 221-234

This study reports MC metabolism by the cytochrome P-450 (CYP) and

glutathione S-transferase (GST) pathways in mouse lung tissue, with

particular reference to the Clara cell. The researchers reached the

following conclusions:

1. Exposure levels of 1000 ppm MC and greater produced increased

levels of DNA synthesis in Clara cells isolated from exposed mice in

culture compared to controls, indicating that these cells are primed

to respond when exposed to MC;

2. A minimum dose of between 1000-2000 ppm of MC is required to

cause vacuolation [the development of cavities] in the Clara cell

when given for 6 hours; and

3. The only biochemical change which correlated with exposure to

MC was lung levels of non-protein sulfhydryl compounds;

Exhibit 120 ``Methylene chloride-induced DNA damage: an interspecies

comparison,'' R.J. Graves, C. Coutts and T. Green, Carcinogenesis, vol.

16 no. 8 pp. 1919-1926, 1995

The researchers measured DNA damage in lung and liver cells from

mice, rats, hamsters and humans. They observed increased DNA single

strand (ss) breaks in mouse liver cells, after exposure to 4000-8000

ppm MC for 6 hours and in mouse lung cells after exposure to 2000-6000

ppm MC. No increase in ss breaks was detected in rat livers after

exposure to 4000 ppm for 6 hours or in rat lungs after exposure to 4000

ppm for 3 hours. Increased numbers of ss breaks were also not detected

in hamster and human liver cells after exposure to MC in vitro at

concentrations up to 90 and 120 mM. In experiments on isolated mouse

Clara cells, the authors observed increased DNA ss breaks in cells

exposed to concentrations of MC of 5 mM and above. According to the

authors, the study suggests that humans, rats and hamsters are

insensitive to MC-induced liver cancer, because those species lack the

high level of GST metabolic activity found in the mouse Clara cell.

Exhibit 121 ``Isolation of two mouse theta glutathione S-transferases

active with methylene chloride, G.W. Mainwaring, J. Nash and T. Green,

Zeneca Central Toxicology Laboratory, 1995.

The researchers used a variety of chromatography methods to isolate

two mouse glutathione S-transferases (MT-1 and MT-2) metabolizing MC,

comparing the observed enzyme activity with that detected in rat GST

(GST 5-5 and GST 12-12). The authors stated as follows:

The difference seen in total methylene chloride metabolizing

activity between rat and mouse in vivo, or in cytosol fractions, is

more than 10 fold which does not appear to be attributable to a

higher specific activity of mouse MT-1 compared to rat GST 5-5. At

present the labile nature of rat GST 12-12 and mouse MT-2 preclude

an assessment of the relative activities of these enzymes in the two

species. However, it seems probable that the higher activity in the

mouse is attributable to greater expression of the one or both

enzymes in that species.

Exhibit 122 ``Mouse Liver glutathione S-Transferase Mediated

Metabolism of Methylene Chloride to a Mutagen in the CHO/HPRT Assay,''

R.J. Graves and T. Green, Zeneca Central Toxicology Laboratory, 1995

This study investigated the mutagenicity of MC in mammalian cells

by inducing mutations at the HPRT locus of CHO cells in mouse livers

through exposure to MC GST metabolites, formaldehyde (a MC metabolite)

and 1,2-dibromoethane (1,2-DBE) (the reference genotoxin).

Based on a comparison of the mutagenic effects of the three

compounds, particularly on the lack of MC-induced DNA-protein cross-

linking in this experimental system, the authors concluded that

formaldehyde does not play a major role in MC mutagenicity.

Accordingly, the researchers viewed the results of this study as

supporting the hypothesis that the DNA ss breaks induced by MC, and the

resultant DNA mutations, are caused by interaction of S-chloromethyl

glutathione with DNA.

Exhibit 123 ``DNA Sequence Analysis of Methylene Chloride-Induced HPRT

Mutations in CHO Cells: Comparison with the Mutation Spectrum Obtained

for 1,2-Dibromethane and Formaldehyde,'' R.J. Graves, P. Trueman, S.

Jones and T. Green, Zeneca Central Toxicology Laboratory, 1995

The researchers compared the spectrum of DNA mutations induced by

exposure to MC with the mutations induced by formaldehyde and 1,2-DBE.

The results provided a spectrum analysis of MC and 1,2-DBE-induced

mutagenesis in mammalian cells and extended the previous observation of

formaldehyde mutagenesis in human lymphoblasts. The results suggested

to the researchers that formaldehyde-induced DNA damage can contribute

to MC mutagenicity, but that the majority of the mutations were derived

from other types of DNA damage, probably via an interaction of S-

chloromethylglutathione with DNA. The researchers noted that a

glutathione conjugate also plays a role in the mutagenicity of 1,2-DBE.

The increases above background mutation frequency detected through this

study were 24.7-fold for 1,2-DBE, 4.7-fold for formaldehyde, and 8-fold

for MC.

Exhibit 124 ``The distribution of glutathione S-transferase 5-5 in the

lungs and livers of mice, rats and humans'' [Preliminary communication,

T. Green, 1995]

This preliminary communication summarizes the results of a study

comparing the distribution of the glutathione S-transferase (GST)

isozyme putatively responsible for metabolizing methylene chloride in

the lungs and livers of mice, rats and humans. The distribution of

enzyme was visualized using oligonucleotide anti-sense probes

complementary to the nucleotide sequences for the transferases. The

results indicated that the GST-specific mRNA could be found in lungs

and livers of all three species. Mouse liver cells (particularly the

nuclei) and mouse lung cells appeared to stain more heavily for the GST

mRNA than the lung or liver cells from rats or humans. Although the

amount of GST-specific mRNA was not quantified in this study, the

authors interpreted the data to suggest that, ``* * * mouse tissues are

stained much more heavily than sections from either rat or human.''

Based on the distribution of the GST mRNA, the author concluded that,

The most significant findings are the presence of very high

concentrations of GST 5-5 mRNA in specific cells and nuclei of mouse

liver and lung. Metabolism of

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methylene chloride at high rates and within nuclei to a reactive but

highly unstable glutathione conjugate is believed to facilitate

alkylation of DNA by this metabolite. The lack of high or nuclear

GST 5-5 concentrations in rat and human tissue, provides an

explanation for the lack of genotoxicity in these species.

II. Public Participation

Comments

Written comments regarding the materials incorporated into the MC

rulemaking record through this notice must be postmarked by November

24, 1995. Four copies of these comments must be submitted to the Docket

Office, Docket No. H-071B, U.S. Department of Labor, Room N-2625, 200

Constitution Avenue, NW., Washington, DC 20210. (202) 219-7894. All

materials submitted will be available for inspection and copying at the

above address. Materials previously submitted to the Docket for this

rulemaking need not be resubmitted.

III. Authority

This document was prepared under the direction of Joseph A. Dear,

Assistant Secretary of Labor for Occupational Safety and Health, U.S.

Department of Labor, 200 Constitution Avenue, NW., Washington, DC

20210.

It is issued under section 6(b) of the Occupational Safety and

Health Act (29 U.S.C. 655), and 29 CFR Part 1911.

Joseph A. Dear,

Assistant Secretary of Labor.

[FR Doc. 95-26228 Filed 10-23-95; 8:45 am]

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