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 Work Assignment Manager 

March 5, 2010 
HPV1 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 22202 
Draft HPV1 Test Rule – Data Adequacy – Repeated-Dose/Repro/Develop Tox CAS No. 409-02-9

PREPARATION OF THE DATA ADEQUACY ASSESSMENT

This document contains the review of a toxicity 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 guides, the study was reviewed for adequacy and 

evaluated for compliance with the test guideline. (The applicable test guideline 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 assigned a Klimisch reliability code. If the study 

was determined to be inadequate, a basis for that conclusion is supplied. For studies already 

assigned Klimisch codes, an explanation is provided if the reviewer’s assessment of data 

adequacy differed from the pre-assigned Klimisch code. 

A detailed evaluation of the test report and study conduct is provided at the end of the 

summary document in a section entitled “Review 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 Procedures, Good Laboratory Practice Standards, and 

Quality Assurance procedures, as well as the use of appropriate controls and test substances. 

Any deficiencies in the descriptions of the study method, test system, animals studied, physical 

conditions, dosage information, individual 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. 

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Draft 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 Heavy 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 with reproductive/developmental toxicity 

screening test 

GLP compliant?: Yes 

Year study performed: 2009 

Test species: Crl:CD (SD rats received from Charles River (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 receipt of rats concurred with order. At the start of treatment 

rats were approximately 11 weeks old and weights 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: F0 females were exposed to a single daily dose of methyl heptanone from 

two weeks before pairing, throughout pairing and gestation, until Day 6 of lactation of the F1 

generation. F0 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. F1 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). 

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

Statistical methods used: Statistical analyses were applied 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 incidence of the finding within the background control population. 

The following sequence of statistical tests was used for sensory reactivity, grip strength and 

motor activity, bodyweight, food consumption, organ 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 compared 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 values >c, as applicable. 

If Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level, 

then parametric analysis was applied. The F1 approximate test was applied. This test is 

designed to detect significant departure from monotonicity of means 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, EMS. 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 distribution with 1 and EDF degrees of freedom. If the F1 approximate 

test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a 

monotonic trend was applied. If the F1 approximate test was significant, suggesting that the 

dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. 

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

were tried. If Bartlett's test was still significant, then non-parametric 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 Shirley's test. The test statistic compares the nonmonotonicity sums of squares, NRSS, for the deviations of the observed mean ranks from the 

maximum likelihood mean ranks with the non-parametric 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 χ2 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 approximate test was significant, suggesting that the 

dose-response was not monotone, Steel's test (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 function and litter as a random effect (Lipsitz et al 1991). Each 

treated group was compared to control using a Wald chi-square test. 

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

(Wilcoxon 1945). 

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

For gestation length an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced 

scores, was applied to all groups. If the test was statistically 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 statistically 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 scored as 5 and irregular, extended and acyclic 

cycles were scored as 6. If the test was statistically 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 statistically significant (p>0.05). If the exact version of the Linear-by-linear test could 

not be calculated (due to the size 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 relationship between organ weight and bodyweight was 

significant at the 10% level (Angervall 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 differences between Control and treated groups were 

expressed at the 5% (p<0.05) or 1% (p<0.01) level. 

Test method/conditions remarks: F0 Females were exposed to a single daily dose of methyl 

heptanone from two weeks before pairing, throughout pairing and gestation, until Day 6 of 

lactation of the F1 generation. F0 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. 

F1 animals were not gavage dosed. 

During the acclimatization period, the animals were checked twice daily for health and general 

condition. Throughout the treatment period, detailed observations were recorded at the following 

times in relation to dose administration: 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 weekly 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 Day 6 of lactation. The 

following measurements, reflexes and responses were 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 ten 6-minute intervals) using a Rodent Activity 

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Draft 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 maternal no observed adverse effect level (NOAEL) 

Reproductive NOAEL (male and female) = 500 mg/kg/day 

Developmental and Offspring 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 killed because of dystocia, with evidence of 

parturition in excess of 24 hours. One female at 500 mg/kg/day was killed on Day 16 of 

gestation with terminal signs that included underactive behavior, irregular 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 administered 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 association with dose administration from Day 3 of study 

were increased salivation with associated chin rubbing and is most likely related to the 

palatability of the formulations, rather than a direct action of the test substance. 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 weight 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, bodyweight gain was essentially similar to 

Controls. During lactation absolute bodyweights for females receiving 500 mg/kg/day were 

significantly low compared with the Controls. Bodyweight gain at 300 and 500 mg/kg/day was 

slightly but not significantly low when compared with the Controls. Bodyweight gain at 100 

mg/kg/day was unaffected by treatment 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 significantly 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/kg/day was unaffected by treatment during the 

pre-pairing period. 

During gestation females receiving 500 mg/kg/day showed marginally low food consumption. 

At 100 or 300 mg/kg/day food consumption was similar to the Controls. 

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

At 500 mg/kg/day food consumption 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 differences that could be attributed to treatment 

with Methyl Heptenone. 

At 500 mg/kg/day there was an increase in the number of females exhibiting 4/5 day cycles or 

irregular cycles. At 100 or 300 mg/kg/day the majority 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-related shift in the distribution of gestation lengths showing 

longer gestation periods compared with Controls. The treatment-related shift may be related to a 

slight delay in fetal growth. The gestation index was unaffected by treatment. Litter size, sex 

ratio, offspring development, survival and 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 incidence of weak tail pinch response during 

Week 5 of treatment. Forelimb grip strength values 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 recording, with a dose relationship being evident. Motor 

activity scores for females on Day 6 of lactation 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 weights. On Day 7 of lactation, adjusted ovarian 

weights for females that received 500 mg/kg/day were high when compared with the Controls. 

At 500 mg/kg/day macroscopic examination at necropsy revealed two out of 10 males with pale 

kidneys and three out 10 with macroscopically 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 aggregations of alveolar macrophages (minimal grade) was observed 

in the lungs of all treated male groups and the high dose female group. Only one male given 500 

mg/kg/day presented a higher severity (slight grade) 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 histopathological examination 

in the liver in all treated male and female groups. These observations are common findings in the 

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

livers of rodents that have been administered xenobiotics and as such are considered to be an 

adaptive change and not a toxic effect of treatment. 

Increased incidence and minimal to moderate severity of cortical tubular 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 incidence of minimal to slight 

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

Thyroid follicular cell hypertrophy (minimal severity) was noted in all treated male groups and 

in females given 300 or 500 mg/kg/day. These changes are not, however, considered of any 

toxicological significance to man. 

Macroscopic examination of F1 offspring that died 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 toxicity, 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 specific changes in thyroid and 

kidney pathology. Despite evidence of systemic effects in the rat at 500 mg/kg/day there was no 

consequential impairment of reproductive function 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 and development was 500 mg/kg/day. 

STUDY RELIABILITY

[2] Reliable with restrictions; the number of corpora lutea is not reported. 

REFERENCE

Angervall, L and Carlstrom, E. (1963) Theoretical criteria for the use of relative organ 

weights and similar ratios in biology. Jnl. Theoret. Biol. 4:254-259 

Bartlett, M.S. (1937) Properties of sufficiency and 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 procedure 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 for Research Workers, 14th edn., p.96. Hafner 

Publishing Company, New York, USA. 

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Huntingdon Life Sciences, Ltd. 2009. Methyl Heptenone Combined Repeated Dose Toxicity 

Study with the Reproduction / Developmental Toxicity Screening Test; EXN0115. 

Lipsitz, S.H., Laird, N.M., and Harrington, D.P., (1991), Generalised Estimating 

Equations for Correlated Binary Data: Using the 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 between treatment means when several dose levels 

are compared with a zero dose control. Biometrics, 27, 103-117. 

Williams, D.A. (1972) The comparison of several dose levels with a zero dose control. 

Biometrics, 28, 519-531. 

8 
Review 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 Screening 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 (TSCA) 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) inspections, their dates, and findings were missing or not signed. 

There is evidence that QA procedures 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 different 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 summary 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/Branch/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 met, 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).