SRC TR-10-071

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT

Methyl Heptanone, CAS No. 409-02-9

Repeated-Dose Toxicity Test With Reproductive/Developmental Screening

Prepared by:

Kendra Moran, Ashlee Aldridge, and Joan D. Garey

Environmental Science Center SRC, Inc.

7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.0.009.004

Submitted to:

OPPT/RAD/HPVCB (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue

Washington, D.C. 20460

Elizabeth Margosches, Project Officer Meena Sonawane, Work Assignment Manager David Brooks, Alternate Wo rk Assignment Manager

March 5, 2010HPV1 Test Rule – Data Adequacy Assessment

Methyl Heptanone, CAS No. 409-02-9

Repeated-Dose Toxicity Test With Reproductive/Developmental Screening

March 2010

Submitted to: High Production Volume Chemicals Branch

Risk Assessment Division

Office of Pollution Prevention and Toxics Environmental Protection Agency

WAM: Meena Sonawane

Alternate WAM: David Brooks 1200 Pennsylvania Avenue, NW

Washington, DC 20460-0001

Prepared By:

SRC, Inc.

Environmental Science Center 2451 Crystal Drive, Suite 804 Arlington, VA 22202Draft HPV1 Test Rule – Data Adequacy – Repeated-Dose/Repro/Develop Tox CAS No. 409-02-9

PREPARATION OF THE DAT A ADEQUACY ASSESSMENT

This document contains the review of a toxi city study conducted in response to the first

HPV TSCA Section 4 Test Rule (71 FR 13708, March 16, 2006). Using the applicable test guideline and professional judgment as guide s, the study was reviewed for adequacy and evaluated for compliance with the test guideline. (T he applicable test guid eline is not always the test guideline required by the test rule, because some test sponsors requested and received approval from EPA to use a different guideline .) The study was summarized according to a standard format, assessed for adequacy, and assi gned a Klimisch reliability code. If the study was determined to be inadequate, a basis for th at conclusion is supplie d. For studies already assigned Klimisch codes, an explanation is prov ided if the reviewer’s assessment of data adequacy differed from the pre-assigned Klimisch code.

A detailed evaluation of the test report a nd study conduct is provided at the end of the summary document in a section entitled “Revie w of TSCA Section 4 Final Study Report”. Any deviations from the applicable test guideline are described. The study is also evaluated for compliance with Standard Operating Procedur es, Good Laboratory Prac tice Standards, and Quality Assurance procedures, as well as the use of appropriate controls and test substances. Any deficiencies in the descri ptions of the study method, test system, animals studied, physical conditions, dosage information, indi vidual animal data, calculations, and statistical analyses are described, and internal inconsistencies within the study report are identified. An overall assessment is provided for each of the following: study conduct, data reliability, and data utility.

1Draft HPV1 Test Rule – Data Adequacy – Repeated-Dose/Repro/Develop Tox CAS No. 409-02-9

REPEATED-DOSE WITH REPRODUCTIVE/DEVELOPMENTAL SCREENING

Title: Repeated-Dose Toxicity Test With Reproductive/Developmental Screening

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Methyl Heptanone, CAS No. 409-02-9

Remarks: The test substance, Baton Rouge MEK Hea vy Ketone Heart Cut C8 Ketone Fraction, was characterized as a complex mixture.

METHOD

Method/guideline followed: Toxic Substances Control Act (TSCA) 15 U.S.C. 2601. 40CFR

799.9365.

Test type: Combined repeated-dose toxicity study w ith reproductive/developmental toxicity screening test

GLP compliant?: Yes

Year study performed: 2009

Test species: Crl:CD (SD rats received from Charles Ri ver (UK) Ltd. Rats were approximately 65 days of age and within a weight range of 325 to 349 g for males and 225 to 249 g for females when ordered. Weight range upon recei pt of rats concurred with orde r. At the start of treatment rats were approximately 11 weeks old and wei ghts were 378 to 448 g for males and 225 to 276 g

for females.

Number of animals/sex/dose: 10/sex/dose

Route of administration: Oral Gavage

Vehicle: Corn Oil

Exposure duration: F 0 females were exposed to a single da ily dose of methyl heptanone from two weeks before pairing, throughout pairing and gestation, until Day 6 of lactation of the F1 generation. F 0 males were exposed to a single daily dos e of methyl heptanone from two weeks before pairing, during pairing and up to te rmination after 5 weeks of treatment. F 1 animals were

not dosed.

Doses/concentrations tested: 0 (control), 100, 300 or 1000 mg/kg/day (The 1000 mg/kg/day dose was reduced to 500 mg/kg/day on Day 3).

2Draft HPV1 Test Rule – Data Adequacy – Repeated-Dose/Repro/Develop Tox CAS No. 409-02-9

Statistical methods used: Statistical analyses were applie d where there was indication of possible meaningful inter-group differences. All statistical analyses were carried out using the individual animal (or litter) as the basic experimental unit. Where applicable, the litter was taken

as the treated unit and the basis for statistical analysis and biological significance was assessed

with relevance to the inci dence of the finding within the background control population. The following sequence of statistical tests was us ed for sensory reactivity, grip strength and motor activity, bodyweight, food consumption, orga n weight and clinical pathology data: If 75% of the data (across all groups) were the same value, for example c, then a frequency analysis was applied. Treatment groups were co mpared using pairwise Fisher's Exact tests (Fisher 1973) for each dose group against the control both for i) values <c versus values >=c, and for ii) values <=c versus va lues >c, as applicable.

If Bartlett's test for variance homogeneity (Bartl ett 1937) was not significa nt at the 1% level, then parametric analysis was applied. The F1 approximate test was applied. This test is designed to detect significant de parture from monotonicity of mean s when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the

observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EM S. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with

standard tables of the F distri bution with 1 and EDF degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not si gnificant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approxima te test was significant , suggesting that the dose response was not monotone, Dunnett's te st (Dunnett 1955, 1964) was performed instead.

If Bartlett's test was significant at the 1% level, then logarithmic and s quare-root transformations

were tried. If Bartlett's test wa s still significant, then non-parametr ic tests were applied. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a nonparametric monotonic trend test, such as Shirle y's test. The test statistic compares the nonmonotonicity sums of squares, NRSS, for the de viations of the observed mean ranks from the maximum likelihood mean ranks with the non-para metric equivalent of the error sums of squares, ERSS (=N*(N+1)/12). The test statistic is H1 = NRSS/ERSS which can be compared to

standard tables of the χ 2 distribution with 1 degree of freedom . If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend (Shirley 1977) was applied. If the H1 appr oximate test was significant, suggesting that the dose-response was not monotone, Steel's te st (Steel 1959) was performed instead.

Pre and post implantation loss and sex ratio were analyzed by generalized mixed linear model with binomial errors, a logit link f unction and litter as a random effect (Lipsitz et al 1991). Each treated group was compared to contro l using a Wald chi-square test.

For resorptions, each treated group was compared to control by exact Wilcoxon rank sum test (Wilcoxon 1945).

3Draft HPV1 Test Rule – Data Adequacy – Repeated-Dose/Repro/Develop Tox CAS No. 409-02-9

For gestation length an exact tw o-tailed Linear-by-linear test (C ytel 1995), with equally spaced scores, was applied to all groups. If the test was st atistically significant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step-down’ process was repeated until the test was no longer st atistically significant (p >0.05). If the exact version of the Linear-bylinear test could not be calculated (due to the size of the table containing the data), then the asymptotic version was used instead.

For oestrous cycles an exact one-tailed (upper-tail) Linear-by- linear test (Cytel 1995) was applied to all groups, using scores appropriate to the severity of the observation assuming 4 day cycles to be normal. The categories were scored as follows: a 4 day cycle was scored as 4, a 4/5 day cycle was scored as 4.5, a 5 day cycle was sc ored as 5 and irregula r, extended and acyclic cycles were scored as 6. If the test was statistically signifi cant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step- down’ process was repeated until the test was no longer statistically signi ficant (p>0.05). If the ex act version of the Linea r-by-linear test could not be calculated (due to the si ze of the table containing the data ), then the asymptotic version

was used instead.

For organ weight data, analysis of covariance was initially performed using terminal bodyweight as covariate. If the within group relati onship between organ we ight and bodyweight was significant at the 10% level (A ngervall and Carlstrom, 1963), then the treatment comparisons were made on adjusted group means in order to allow for differences in bodyweight which might influence the organ weights. Significant differen ces between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Test method/conditions remarks: F 0 Females were exposed to a single daily dose of methyl heptanone from two weeks before pairing, thr oughout pairing and gestation, until Day 6 of lactation of the F1 generation. F 0 Males were exposed to a single daily dose of methyl heptanone from two weeks before pairing, during pairing and up to termination after 5 weeks of treatment.

F 1 animals were not gavage dosed.

During the acclimatization period, the animals were checked twice daily fo r health and general condition. Throughout the treatment period, detailed obs ervations were recorded at the following

times in relation to dose administ ration: 1) pre-dose observation; 2) as each animal was returned to its home cage; 3) at the end of dosing of each group; 4) between one and two hours after completion of dosing of all groups; and 5) as late as possible in the working day. Observations of males occurred daily through Week 1, twice we ekly in Week 2, and weekly thereafter. Observations of females occurred daily through Week 1, twice weekly in Week 2 and weekly until mated in Week 3. During gestation this took place on Days 1, 6, 12, 18 and 20 and on Days

2 and 6 of lactation.

Sensory reactivity and grip strength assessments were performed (before dosing) on all male animals during Week 5 of treatment and all surviving females on Da y 6 of lactation. The following measurements, reflexes and responses we re recorded on all male animals during Week 5 of treatment and all surviving females on Day 6 of lactation: Approach response, Sensory reactivity, Grip strength, Touch response, Auditory startle reflex, Tail pinch response, and Motor activity (counting infra-red beam breaks over te n 6-minute intervals) us ing a Rodent Activity

4Draft HPV1 Test Rule – Data Adequacy – Repeated-Dose/Repro/Develop Tox CAS No. 409-02-9

Monitoring System.

RESULTS

Endpoint value(s): Maternal NOAEL = 100 mg/kg/day was the matern al no observed adverse effect level (NOAEL) Reproductive NOAEL (male and female) = 500 mg/kg/day Developmental and Offspr ing NOAEL = 500 mg/kg/day

Results remarks: One female at 300 mg/kg/day and one female at 500 mg/kg/day were sacrificed early because they failed to litter. One female at 500 mg/kg/day was sacrificed following litter death. One control female was k illed because of dystocia, with evidence of parturition in excess of 24 hours. One fema le at 500 mg/kg/day was killed on Day 16 of gestation with terminal signs that included unde ractive behavior, irregu lar respiration and a discharge of blood from the vagina. One Control male was found dead in the cage three days

before the terminal sacrifice.

Initially a high dose of 1000 mg/kg/day was ad ministered and animals were immediately underactive, had partially closed eyelids, piloerection, hunched posture, reduced body tone and prostration for up to 4 hours. From Day 3 on, the high dose was reduced to 500 mg/kg/day and

there was an immediate reduction in acute effects.

The predominant signs observed in associati on with dose administration from Day 3 of study

were increased salivation with associated ch in rubbing and is most likely related to the palatability of the formulations, rather than a direct action of the test subs tance. Detailed physical

examinations and arena observations did not reveal any signs that could be related to treatment with Methyl Heptenone.

Both males and females at the highest dose showed mean weight loss for the first four days and

remained lower than controls as their we ight gain increased through the study. Overall bodyweight gain for females at 500 mg/kg/day was slightly low when compared with Controls. For females receiving 100 or 300 mg/kg/day, bod yweight gain was essentially similar to Controls. During lactation absolute bodyweight s for females receivi ng 500 mg/kg/day were significantly low compared with the Controls. Bodyweight ga in at 300 and 500 mg/kg/day was slightly but not significantly low when compar ed with the Controls. Bodyweight gain at 100 mg/kg/day was unaffected by treatm ent during the lactation phase.

Males and females that initially received 1000 mg/kg/day on Days 1 and 2 of study and subsequently received 500 mg/kg/day, had signi ficantly low food consumption during Days 1 to 3 of treatment. From Day 4 to Day 15 of the pre-pairing period food consumption values for both males and females at 500 mg/kg/day showed recovery and were essentially similar to Controls. Food consumption at 100 or 300 mg/k g/day was unaffected by treatment during the pre-pairing period.

During gestation females receiving 500 mg/kg/da y showed marginally low food consumption. At 100 or 300 mg/kg/day food consump tion was similar to the Controls.

5Draft HPV1 Test Rule – Data Adequacy – Repeated-Dose/Repro/Develop Tox CAS No. 409-02-9

At 500 mg/kg/day food consumpti on was significantly low during lactation (Days 1-7) and at 300 mg/kg/day consumption was low during Days 1-3 of lactation (p<0.05). Food consumption at 100 mg/kg/day was unaffected by treatment.

Hematological and blood chemistry investigations for males after 5 weeks of treatment and for females on Day 7 of lactation did not reveal any di fferences that could be attributed to treatment with Methyl Heptenone.

At 500 mg/kg/day there was an increase in the nu mber of females exhibiting 4/5 day cycles or irregular cycles. At 100 or 300 mg/kg/day the major ity of animals exhibited regular 4-day cycles. This effect on oestrous cycles was therefore not considered to represent an adverse effect of

treatment.

The pre-coital interval, mating performance and fertility were unaffected by treatment. At 500 mg/kg/day there was a treatment-re lated shift in the distribution of gestation lengths showing longer gestation periods compared with Controls. The treatment-rela ted shift may be related to a slight delay in fetal growth. The gestation i ndex was unaffected by treatm ent. Litter size, sex ratio, offspring development, survival a nd bodyweight performance were unaffected by treatment with Methyl Heptenone at dose levels up to 500 mg/kg/day.

Males receiving 500 mg/kg/day showed a high in cidence of weak tail pinch response during Week 5 of treatment. Forelimb grip strength valu es for females receiving 500 mg/kg/day were low on Day 6 of lactation, but hind limb values were unaffected. During Week 5 of treatment, low beam scores (cage floor activity) for males in all treated groups were low, compared with Controls, during the first 6 minutes of recordi ng, with a dose relationshi p being evident. Motor activity scores for females on Day 6 of lactat ion showed considerable inter and intra-group

variation but there was no evidence of an effect of treatment.

After 5 weeks of treatment treated males showed significantly high adjusted kidney weights at all dose levels (a dose response was apparent) and males that received 300 or 500 mg/kg/day also showed significantly high adjusted liver weight s. On Day 7 of lact ation, adjusted ovarian weights for females that receive d 500 mg/kg/day were high when co mpared with the Controls.

At 500 mg/kg/day macroscopic exam ination at necropsy revealed tw o out of 10 males with pale kidneys and three out 10 with m acroscopically enlarged livers. Macroscopic examination of females on Day 7 of lactation did not reveal any findings that could be attributed to treatment.

An increased incidence of aggr egations of alveolar macrophage s (minimal grade) was observed in the lungs of all tr eated male groups and the high dose female group. Only one male given 500 mg/kg/day presented a higher severity (slight gr ade) than Controls. The increase in incidence was slight and a dose response was not apparent, this finding was therefore considered not to be of any toxicological significance.

Slight hepatocyte centrilobular hypertrophy was observed during histopat hological examination in the liver in all treated male and female gr oups. These observations are common findings in the

6Draft HPV1 Test Rule – Data Adequacy – Repeated-Dose/Repro/Develop Tox CAS No. 409-02-9

livers of rodents that have been administered xe nobiotics and as such ar e considered to be an adaptive change and not a t oxic effect of treatment.

Increased incidence and minimal to moderate se verity of cortical t ubular hyaline droplets deposition, tubular granular casts and cortical tubular basophilia were observed in the kidney in all treated male groups, compared with Controls. An increased in cidence of minimal to slight

interstitial inflammation was seen in all treated male gr oups, compared with Controls.

Thyroid follicular cell hypertr ophy (minimal severity) was noted in all treated male groups and in females given 300 or 500 mg/kg/day. These ch anges are not, however , considered of any toxicological significance to man.

Macroscopic examination of F1 offspring that di ed prematurely or were killed at scheduled termination on Day 7 of age did not reveal any findings that could be attributed to treatment.

CONCLUSIONS It was concluded in this study that in terms of general t oxicity, 100 mg/kg/day was the no

observed adverse effect level (NOAEL), based on bodyweight and food consumption decreases, as well as adaptive changes in the liver morphology and rat spec ific changes in thyroid and kidney pathology. Despite evidence of systemic e ffects in the rat at 500 mg/kg/day there was no consequential impairment of reproductive f unction and the high dose of 500 mg/kg/day was considered to be the NOAEL in terms of reproductive function and fertility. The NOAEL in terms of offspring survival a nd development was 500 mg/kg/day.

STUDY RELIABILITY [2] Reliable with restrictions; the numb er of corpora lutea is not reported.

REFERENCE Angervall, L and Carlstrom, E. (1963) Theore tical criteria for the us e of relative organ weights and similar ratios in biolo gy. Jnl. Theoret. Biol. 4:254-259

Bartlett, M.S. (1937) Properties of sufficiency a nd statistical tests. Proceedings of the Royal Society. Series A, 160, 268-282.

Cytel (1995) StatXact 3 for Windows: Statistical Software for Exact Nonparametric Inference. Cytel Software Corporation, NC, USA.

Dunnett, C.W. (1955) A multiple comparison pro cedure for comparing several treatments with

a control. Journal of the American Statistical Association, 50, 1096-1121.

Dunnett, C.W. (1964) New tables for multiple comparisons with a control. Biometrics, 20,

482-491.

Fisher, R.A. (1973) Statistical Methods fo r Research Workers, 14th edn., p.96. Hafner Publishing Company, New York, USA.

7Draft HPV1 Test Rule – Data Adequacy – Repeated-Dose/Repro/Develop Tox CAS No. 409-02-9

Huntingdon Life Sciences, Ltd. 2009. Methyl Hept enone Combined Repeated Dose Toxicity Study with the Reproduction / Developmen tal Toxicity Screening Test; EXN0115.

Lipsitz, S.H., Laird, N.M., and Harring ton, D.P., (1991), Generalised Estimating Equations for Correlated Binary Data: Using th e Odds Ratio as a Measure of Association, Biometrika, 78, 153-160.

Shirley, E.A.C. (1977) A non-parametric equivalent of Williams' test for contrasting increasing dose levels of a treatment. Biometrics, 33, 386-389.

Steel, R.G.D. (1959) A multiple comparison rank sum test: treatments versus control, Biometrics, 15, 560-572.

Wilcoxon, F. (1945) Individual comparisons by ranking methods. Biometrics Bulletin, 1,

80-83.

Williams, D.A. (1971) A test for differences betw een treatment means when several dose levels are compared with a zero dose control. Biometrics, 27, 103-117.

Williams, D.A. (1972) The comparison of severa l dose levels with a zero dose control. Biometrics, 28, 519-531.

8Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman

Chemical Name: Methyl Heptanone

CAS Registry Number: 409-02-9

Report Title: Reproductive Developmental Toxicity Screenin g Test by Oral Gavage Administration to CD Rats

Test Sponsor: Exxon Mobil Chemical Company

Testing Lab: Huntingdon Life Sciences Ltd.

Test Standard: Toxic Substances Control Act (T SCA) 15 U.S.C. 2601. 40 CFR 799.9365.

Date Study Received by EPA: 6/23/09 (date posted to http://www.regulations.gov )

Date Study Due to EPA: 5/17/07

Special Instructions (e.g., regarding approved protocol modifications, etc.):

Part I. Test Report and Study Conduct

Enter one of the following codes into each of the checkboxes 1 through 13: Agree [ A ], Disagree [ D ], Not Applicable [ NA ], or Cannot Determine [ C ], and add comment, below, as needed

[D] 1. The final study report indicates evidence of gross, unauthorized or undocumented deviation from the EPA test standard requirements, including “shall,” “must,” or “will” violations. List significant test standard deviations and “shall,” “must,” or “will” violations in comments below.

[D] 2. The final study report indicates evidence of deviations from the “should” statements in the study protocol that may have a bearing on the study conclusions. List these in comments below.

[D] 3. Approved modifications of test standard or testing protocol (CITB PM describe or attach) were not followed or are inadequately documented.

[D] 4. There is evidence that laboratory Standard Operating Procedures (SOPs) were inadequately followed.

[D] 5. The Statement of Quality Assurance (QA) inspect ions, their dates, and findings were missing or not signed. There is evidence that QA pr ocedures may be inadequate.

[D] 6. There is evidence that Good Laboratory Practice Standards (GLPS) were inadequately followed.

[D] 7. The test, control, or reference substances are di fferent from that specified in the section 4 test rule or

Enforceable Consent Agreement (ECA).

[D] 8. Animal identification is inadequate or unclear; use of unhealthy animals or other factors (e.g., deviations from temperature, lighting, dosing) are apparent.

[D] 9. The final study report inadequately describes the study method.

[D] 10. The final study report inadequately describes the test system.[D] 11. The final study report inadequately describes the dosage information.

[D] 12. The final study report does not accurately reflect the individual animal data (or that of other discrete observations pertinent to the type of study conducted), or individual data were not provided.

[D] 13. The final study report inadequately describes the calculations, interpolations, data transformations, statistical analyses, or modeling parameters used.

Part II. Conclusions

Place a check mark beside the summar y statement(s) that apply to this review and add comments below as appropriate

Part A. To be filled out by Reviewer

Study Conduct 1. The study was well-conducted. [x] 2. The study was generally well-conducted, with some minor [ ] deficiencies (list in notes below). 3. The study conduct was significantly deficient or questionable, (give [ ] reasons in notes below).

Data Reliability 4. The study results are precise and accurate. [x] 5. Appropriate methods were used to determine [x] statistical significance. 6. If repeated, the test results would be expected to be the [x]

same.

Data Utility 7. The data are useful for risk assessment for the endpoint tested. [x] 8. The data are useful for some other purpose (e.g., dose-setting). [ ] 9. The data are not useful for any purpose (describe in notes below). [ ]

Reviewer Comments (continue on separate page, if needed):

Reviewer Name/Title/Bran ch/Division/Office: Kendra Moran, SRC, Inc.

Reviewer Signature: David Brooks/HPVCB/RAD/OPPTS

Part B. To be filled out by CITB PM

Tiered Testing Decision 1. The tiered testing trigger was me t, additional testing is indicated. [ ] (when applicable) 2. The trigger for additional (tiered) testing was not met. [ ]

Study Acceptability 3. The study is acceptable. [ ] 4. The study may not be acceptable [ ] (provide rationale in memo to CITB chief).