tPA/600/1-91/002
June 1991
2-METHYLHEXANOIC ACID
DEVELOPMENTAL TOXICITY TESTING
by
Michael G. Narotsky
ManTech Environmental Technology, Inc.
Research Triangle Park, NC 27709
Contract No. 68-02-4450
Robert J. Kavlock
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27709
May 1, 1991
Abstract: As part of an investigation of the developmental effects and struc-
ture-activity relationships of aliphatic acids, 2 -methylhexanoic acid was
administered by gavage to Sprague-Dawley rats on gestation days 6-15 at doses
of 0, 300, and 400 mg/kg/day. The dams were allowed to deliver and their lit-
ters were examined through postnatal day 6. Pups that were found dead were
examined for soft-tissue alterations. On day 6, two survivors per litter were
preserved for skeletal examination. Maternal toxicity was demonstrated at
both 300 and 400 mg/kg by weight loss and altered respiration (rales,
dyspnea). In spite of the maternal toxicity present, there were no clear
toxic effects on development; litter size, pre- and postnatal viability, and
pup weights were unaffected by treatment. Skeletal examinations of selected
pups yielded inconclusive data; a slight increase in the incidence of lumbar
ribs was present at 400 mg/kg, but was not clearly attributable to treatment.
This document is intended for internal Agency use only. Mention of trade
names or commercial products does not constitute endorsement or recommendation
for use.

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DEVELOPMENTAL TOXICITY TESTING
2-METHYLHEXANOIC ACID
INTRODUCTION
Valproic acid (VPA), an anticonvulsant, is teratogenic in humans and rodents
(Lanuner et al. , '87), We are using the rat to investigate the developmental
effects and strueture-activity relationships of VPA and related chemicals.
Studies with VPA, butanoic, 2-ethylbutanoic, pentanoic, 2-methylpentanoic, 2-
ethylhexanoic, 3-methylhexanoic, 5-methylhexanolc, octanoic, and 2-nethyloc-
tanoic acids were reported previously (Narotsky et al., '88, '89a, '89b;
Mervin et al., '89a, '89b, 'S9c; Narotsky and Kavlock, '89, '90a, '90b, '91).
In this effort, we investigated 2-methylhexanoic acid (2MH) using an in vivo
developmental toxicity screen developed by Chernoff and Kavlock ('82).
MATERIALS AND METHODS
Chemical
The test compound was 2-methylhexanoic acid (99%, Aldrich Chemical Co., Mil-
waukee, WI, Lot No. 03612EV). Corn oil (laboratory grade, Fisher Scientific,
Co.) was used as the vehicle. Solutions were prepared at appropriate concen-
trations to provide the desired dose when administered at 1 ml solution/kg
body weight.
Animal Husbandry
Timed pregnant 90-day-old Sprague-Dawley rats, 240-280 grams, were obtained
from Charles River Laboratories, Raleigh, NC and individually housed in
18x9hx8" polycarbonate cages. Heat-treated wood shavings were supplied for
bedding. The animals were provided feed (Purina Laboratory Rodent Chow
#5001) and tap water ad libitum and a 12-hr. photoperiod (lights on at 0600).
Room temperature and relative humidity were maintained at 72+2°F and 50+10%,
respectively.
Procedures
Dose-Ranee-Finding Study;
In order to determine appropriate dose levels for the present study, a prelim-
inary study was conducted using nongravid females of the same age and strain.
Six females, individually housed, were assigned to each of five groups receiv-
ing 0, 300, 600, 900, or 1200 mg 2MH/kg/day for 10 days; dosing solutions were
administered by gavage at 2 ml/kg initial body weight. Animals were main-
tained for 5 days posttreatment. Body weights were determined on study days
1, 3, 5, 8, 11, and 15 and clinical observations were recorded.
Developmental Toxicity Screen:
The day that evidence of mating (e.g., sperm in the vaginal smear) was detect-
ed was designated gestation day (CD) 0. On GD 3, the rats were assigned to
one of three treatment groups using a nonbiased procedure that assured a
homogeneous distribution of body weights among groups. Twenty, 15, and 15
rats were gavaged daily on GD 6-15 at dosages of 0, 300, and 400 mg 2MH/kg,
respectively, at a volume of 1 ml solution/kg body weight. All doses were
based on individual GD-6 body weights. Maternal body weights were determined
on GD 3, 6, 8, 10, 13, 16, and 20 and the rats were examined throughout the
experimental period for clinical signs of toxicity.
2

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i
DEVELOPMENTAL TOXICITY TESTING
2-METHYLHEXANOIC ACID
Beginning on GD 20, the dams were observed periodically (up to seven times per
day) to determine the stage (completed, in progress, or first pup delivered)
and time of parturition. Postnatal day (PD) 1 was defined as GD 22 independ-
ent of the actual time of parturition. Pups in each litter were counted on PD
1, 3, and 6 and were weighed collectively on PD 1 and 6. Pups that were found
dead with no gross malformations were preserved in Bodian's solution (2%
formaldehyde, 5% acetic acid, 72% ethanol) and examined for soft-tissue alter-
ations. On PD 6, two survivors (one/sex, if possible) from each litter were
fixed in 65% ethanol and stained with alizarin red S for skeletal examination.
Soft-tissue examinations included examination of the head vising free-hand
sections similar to that described by Wilson ('65). The thoracic and abdomi-
nal viscera were examined by dissection. After PD 6, the dams were killed and
uterine implantation sites were counted. Females that did not deliver were
killed on presumed GD 24 and their uteri were examined to determine pregnancy
S tcltVIS .
Statistics
Data were analyzed using the General Linear Models procedure on SAS. Since
the a priori hypothesis was that treatment could only reduce the number of
surviving progeny, one-tailed tests were used for pertinent data. Gestation-
length data were analyzed using the Kruskal-Wallis test; ranks were based on
the time and stage that parturition was observed. The number of live FD-1
pups was used as a covariate in the analysis of pup weights. Similarly, the
number of implants was used as a covariate in the analyses of the number of
live pups. When a significant treatment effect was detected by analysis of
variance, Student's t-test on least-squares means was used to identify indi-
vidual groups that were significantly different from the control group.
Prenatal loss for each litter was defined as the number of implants.that were
not viable at the PD-I examination. Postnatal loss was defined as the number
of implants that were viable on PD 1, but not on PD 6. An additional parame-
ter, perinatal loss, was defined as the number of implants that did not sur-
vive to PD 6.
Dams with only one implant were excluded from statistical analyses. Fully
resorbed litters were regarded to have zero live pups at all postnatal exami-
nations. Litters that were delivered after PD 1 were included in the PD-3,
PD-6, and perinatal loss analyses, but not in the PD-1, pre-, and postnatal
loss analyses. Pup-examination data were considered anecdotal and were not
statistically analyzed.
RESULTS
Dose-Ranee-Findiny Study
Body weight losses were evident after two doses at 1200 tog/kg and after seven
doses at 300 and 600 rag/kg (Table 1). Evaluation of body weight patterns at
900 mg/kg was confounded by high mortality in this group. Signs of respirato-
ry toxicity (usually rales) were present in all animals treated with 2MH.
3

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DEVELOPMENTAL TOXICITY TESTING
2-METHYLHEXANOIC ACID
Dyspnea was evident in one female in each of the groups receiving 600, 900,
and 1200 rag/kg. Motor depression (ataxia or decreased motor activity) was
observed in two females at 600 mg/kg and three females at 1200 mg/kg. Death
occurred in one 300-mg/kg female (day 8), In three females at 900 mg/kg (days
2, 3, and 5), and in one female at 1200 mg/kg (day 4), All animals survived
at 600 mg/kg.
Based on the results of this dose-range-finding study, dose levels of 0, 300,
and 400 mg/kg were selected for the developmental toxicity screen.
Maternal Data
All females survived the experimental period; however, maternal toxicity was
demonstrated by signs of respiratory toxicity (dyspnea, rales, vocalization,
nasal congestion) in most females of both dose groups (Table 2, Appendix 1).
Ataxia was observed in two (13%) high-dose females within four hours after
dosing on the ninth day of treatment.
Body weight losses after the first 2 days of dosing were significant at both
dose levels (Table 2, Appendix 2). Mean body weights decreased 0.5 and
10.3 g. for the low- and high-dose groups, respectively; whereas control dams
gained 9.8 g. over the first 2 days of treatment. Significant dose-related
reductions in weight gain were evident in both dose groups after 4 doses and
on GD 20. Gestational weight gains adjusted for live-litter weights were also
affected in a dose-related manner and were significantly reduced in the high-
dose group.
Developmental Data
Gestation lengths, litter sizes, pre- and postnatal viability, and pup weights
were comparable in all groups (Table 3, Appendix 3). Perinatal mortality was
marginally increased in the low-dose group due to the death of one litter
(#2033) with four pups. High-dose values for perinatal mortality were com-
parable to controls.
Visceral examinations were conducted on two pups from low-dose litter #2033.
Both pups, killed moribund on PD 3, had a dilated ureter; one pup also had a
dilated renal pelvis. The remaining two pups in this litter died after PD 3
and were apparently cannibalized. An additional pup in each of the control
and high-dose groups was found dead, but autolysis precluded a complete exami-
nation of these specimens. Skeletal examinations conducted on two PD-6 survi-
vors from each litter revealed slightly increased incidences of lumbar ribs in
the high-dose group (Table 3). Virtually all the lumbar ribs were rudimentary
(i.e., focal). The only exception was a short rib (less than one-half the
length of the adjacent rib) observed in a high-dose pup.
DISCUSSION
Maternal toxicity was demonstrated at both 300 and 400 mg/kg by weight loss
early in the treatment period and by respiratory effects. Isolated cases of
ataxia and reduced gestational weight gains adjusted for PD-1 litter weights
4

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I
DEVELOPMENTAL TOXICITY TESTING
2-METHYLHEXANOIC ACID
Despite these maternal effects, no clear developmental toxicity was evident at
either dose level. There were no delays in parturition, nor any definitive
effects on progeny growth or viability. A marginal increase in perinatal mor-
tality at 300 mg/kg was attributed to the postnatal death of one litter
(#2033) with four pups. The cause of this litter's death was undetermined;
but, due to the nondose-related pattern, we regard this finding to be unrelat-
ed to treatment.
A slight increase in the incidence of lumbar ribs in selected FD-6 pups at
400 mg/kg suggested a developmental effect similar to that of VPA and 2-ethyl-
hexanoic acid (2EH). VPA, in a Segment II study (Narotsky et al., '88), and
2EH, in an in vivo screen (Narotsky et al., '89a), both induced lumbar ribs.
However, unlike the present study, full-length lumbar ribs were observed. In
addition, VPA and 2EH were also associated with cervical ribs, fused ribs,
fused vertebrae, extra presacral vertebrae, and missing caudal vertebrae.
Since only a small number of randomly selected pups from each litter were
examined in the present study, and no other evidence of developmental toxicity
was present at this dose level, these skeletal data were considered inconclu-
sive .
In a comparison of structure-activity relationships of 2MH and the ten other
aliphatic acids studied in our laboratory thus far (Narotsky et al., '88,
'89a, '89b; Mervin et al., '89a, '89b, '89c; Narotsky and Kavlock, '89, '90a,
'90b, '91), all induced maternal respiratory toxicity. However, only 3-raeth-
ylhexanoic acid, 2EH, and VPA (2-propylpentanoic acid) caused pronounced motor
depression (e.g., ataxia, lethargy) in the developmental toxicity screen. 2MH
and 2-methyloctanoic acid also caused motor depression, but with much lower
incidence. Only VPA and 2EH were associated with dramatic effects on devel-
opment; these two compounds caused delayed parturition in addition to malfor-
mations of the vertebrae and ribs (see above). Structurally, 2MH differs from
the developmentally toxic 2EH, only by possessing a one-carbon side chain.
Thus, the lack of developmental toxicity for 2MH demonstrates the strict
structural restraints for the biological activities for these congeners.
A qualitative summary of results obtained in this project to date is presented
in Table 4. Additional testing with other aliphatic acids as well as halogen-
and alkyloxy-substituted compounds will be conducted to gain further insights
in the structure-activity relationships of this class of chemicals and their
effects on development.
ACKNOWLEDGEMENT
We gratefully acknowledge the excellent technical assistance of Bonnie Hamby.
5

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DEVELOPMENTAL TOXICITY TESTING	2-METHYLHEXAN01C ACID
REFERENCES
Chernoff, N, arid R.J. Kavlock. (1982) An in vivo teratoLogy screen utilizing
pregnant mice. J. Toxicol. Environ. Health, 10:541-550.
Hervin, S.J., M.G. Narotsky, and R.J. Kavlock. (1989a) 2-Methyloctanoic acid,
developmental toxicity testing. Unpublished report.
Mervin, S.J., M.G. Narotsky, and R.J. Kavlock. (1989b) Octanoic acid, develop-
mental toxicity testing. Unpublished report.
Mervin, S.J., M.G. Narotsky, and R.J. Kavlock. (1989c) 2-Ethylbutanoic acid,
developmental toxicity testing. Unpublished report.
Narotsky, M.G., V.M. Boncek, and R.J. Kavlock. (1988) 2-Propylpentanoic acid
(valproic acid), developmental toxicity testing. Unpublished report.
Narotsky, M.G. and R.J. Kavlock (1989) Pentanoic acid, developmental toxicity
testing. Unpublished report.
Narotsky, M.G. and R.J. Kavlock (1990a) 3-Methylhexanoic acid, developmental
toxicity testing. Unpublished report.
Narotsky, M.G. and R.J. Kavlock (1990b) 5 -Me thylhexanolc acid, developmental
toxicity testing. Unpublished report.
Narotsky, M.G. and R.J. Kavlock (1991) 2-Methylpentanoic acid, developmental
toxicity testing. Unpublished report.
Narotsky, M.G., S.J. Mervin, and R.J. Kavlock. (1989a) 2-Ethylhexanoic acid,
developmental toxicity testing. Unpublished report.
Narotsky, M.G., S.J, Mervin, and R.J. Kavlock, (1989b) Butanoic acid, develop-
mental toxicity testing. Unpublished report.
Wilson, J.G. (1965) Methods for administering agents and detecting malforma-
tions in experimental animals. In: Teratology: Principles and Techniques.
J.G. Wilson and J. Warkany, eds. Univ. Chicago Press, Chicago, pp. 262-277,
6

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Table 1: Summary of Dose-Range-Finding Study of Nongravid Rats Gavaged for 10 Days
With 2-Methylhexanoic Acid.
DOSE LEVEL (mg/kg/day)
0	300	600	900	1200
No. Rats











Treated
6

6


6


6

6
Affected











Rales
0

5


5


6

5
Dyspnea
0

0


1


1

1
Ataxia
0

0


1


0

2
Dec. Activity 0

0


1


0

1
Death
0

1


0


3

1
Mean ± S.E. (n)
Body Weights (g)










Day 1
273.7 ± 3.9(6)
273.0
+
4.8(6)
270.8
+
3.1(6)
275.8
± 6.7(5)a
273.7
± 6.7(6)
Day 3
275.7 ± 3.8(6)
276.5
+
4.5(6)
272.8
+
5.2(6)
276.6
±12.1(5)
267.3
±10.2(6)
Day 5
279.3 ± 4.8(6)
279.5
+
4.5(6)
275.5
+
3.9(6)
276.0
± 7.6(4)
267.6
±13.6(6)
Day 8
282.8 ± 6.4(6)
277.8
+
3.8(6)
270.8
+
5.0(6)
279.0
±10.7(3)
268.4
±13.4(5)
Day 11
284.7 ± 7.1(6)
287.4
+
5.7(5)
275.8
+
5.1(6)
282.0
±18.2(3)
263.2
±10.6(5)
Day 15
294.2 ± 7.8(6)
292.8
+
5.0(5)
289.7
+
4.4(6)
306.3
±19.4(3)
278.8
± 8.9(5)
a Animal that died prior to Day 3 was excluded from the calculations.

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Table 2: Maternal Data for Rats Gavaged With 2 -Me thylhexano i c Acid
on Gestation Days (GD) 6-15.
DOSE LEVEL (mg/kg/day)
0	300	400
No. Females
Treated

20

15

15

Affected <
(%)









Dyspnea


0


1
( 7)

1
( 7)
Rales


1

13
(87)
15
(100)
Vocalization

0


5
(33)

9
(60)
Ataxia


0


0


2
(13)
Died


0


0


0

With 1 Implant

0


0


0

Remaining
Pregnant
17

11

13

vernal Body
Weights (g)









Day 6

278.2
+
2.0
280.1
+
3.8
283.7
+
2.4
Day 8

288.1
+
2.3
279.6
+
6.7
273.4
+
5. 7a
Day 10

300.3
+
2.2
287.8
+
7. 5a
289.9
+
3. 8a
Day 13

321.8
+
2.7
308. 7
+
6. 8a
308.9
+
4. 6a
Day 16

351.6
+
3.1
333.1
+
7.1*
327.7
+
7.6**
Day 20

414.3
+
4.6
394.1
+
5.9*
389.8
+
8.2*
:ernal Weight Gains (g)









Days 6 - 8

9.8
+
1.3
-0.5
+
4.1*
-10.3
+
5.0***
Days 6-10

22.1
+
1.7
7.7
+
5. 6**
6.2
+
3.2**
Days 10-16

51.3
+
2.2
45.3
+
3.6
37.8

7.2
Days 16-20

62.7
+
2.1
61.0
+
3.4
62.2
+
2.3
Days 6-16

73.4
+
2.5
53.0
+
4.5**
44.0
+
7,6***
Days 6-20

136.1
+
4.0
114.0
+
4.3*
106.2
+
8. 3**
Days 6-20
(adj e)
52.7
+
3.6
41.2
+
4.9
31.6
+
6.8*
a Marginally significant difference from control value (p _ 0.10).
* Significantly different from control value (p _ 0.05).
** Significantly different from control value (p _ 0.01).
*** Significantly different from control value (p _ 0.001).
e Adjusted for the live-litter weight on postnatal day 1. The sample
size is the number of dams with live pups on postnatal day 1
(see Table 3).
i Mean + S.E. using the number of females "remaining pregnant" as the
sample size.

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Table 3: Developmental Data for Rats Gavaged With 2-Methylhexanoic Acid
on Gestation Days 6-15.
DOSE LEVEL (mgAg/day)

0

300

400

No. Dams
17

11

13

Delivering






Gestation Day 21
16

10

11

Gestation Day 22
1

1

2

With Live Pups: Day 1
17

11

13k
Day 6
17

10

12
No. Implants^
13.0 +
0.41
11.5 +
0.9
12.6 +
0.6
No. Live Pups: Day 1.
11.8 +
0.4
9.9 +
1.0
10.8 +
0.6
Day 3^
11.8 +
0.4
9,9 +
1.0
10.7 +
0.6
Day
11.7 +
0.4
9.5 +
1.3
10.7 +
0.7
Percent Loss: Prenatal
9.3 +
2.1
14.2 +
4.5
14,2 +
3.4
Postnatal^
0.5 +
0.5
9.1 +
9.1
0.6 +
o.&|
Perinatal^
9.8 +
2.0
23.2 +
8. 8a
14.6 +
3.6
Perinatal Mortality (No.)J
1.3 +
0.3
2.0 +
0.5
1.9 +
0.5'
Pup Weight (g): Day lm
7.1 +
0.1
7.5 +
0.3
7.0 +
0.2,
Day 6
14.1 +
0.3
15.4 +
0.8
14.0 +
0.6
Skeletal Findings0:
No, Pups (Litters)
Examined	34 (17)	20 (10)	26 (13)
Affected
Rudimentary Lumbar Rib 8 (6)	4 (4)	11 (7)
Extra Sternebra	4 (4)	1	0
Percent Pups (Litters)
Affected
Rudimentary Lumbar Rib 24 (35)	20 (40)	42 (54)
Extra Sternebra	12 (24)	5 (10)	0
Mean + S.E. Percent/Litter
Rudimentary Lumbar Rib 23.5 + 8.7 20.0+8.2 42,3+12.5
Extra Sternebra	11.8+5.3	5.0+5.0	0
a Marginal significant difference from control value (p _ 0.10).
b Day-6 data from Dam 2020 excluded from calculations due to
malfunctioning water spigot,
i Mean + S.E.
j Sample size: the total number of dams (I.e., 17, 10, and 13 for
the control, low-, and high-dose groups, respectively),
k Sample size: the number of litters examined on Day 1 (i.e., 17, 11,
and 13 for the control, low-, and high-dose groups, respectively),
m Sample size: the number of dams with live pups on the day specified,
o Two PD-6 pups were examined from each litter.

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Table 4: Summary of Results of Developmental Toxicity Assays
With Aliphatic Acids.
Aliphatic
Acid
Maternal Effects
Altered
Motor Respi-
Depression ration
Developmental Effects
Delayed Reduced Decreased
Weight Partu-
Loss rition
Pup	Pup	Maifor-
Weight Viability mations
Butanoic
2-Ethyl-
butanoic
+
+
+
Pentanoic
2-Methyl-
pentanoic
2-Propyl-
pentanoic6
+
+
+
+
2-Ethyl-
hexanoic
2-Methyl-	+	+	+
hexanoic
3-Methyl-	+	+	+
hexanoic
5-Methyl-	+	+
hexanoic
Octanoic	+	+	+	+
2-Methyl-	+	+	+	-	-
octanoic
a Occurred only in dams with peripartuo respiratory symptoms,
b Positive in Segment II study, negative in screen,
d In dose-range-finding study,
e Results of segment II study included.

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APPENDIX 1
2-METHYLHEXANOIC ACID
DEVELOPMENTAL TOXICITY TESTING IN SPRAGUE-DAWLEY RATS
INDIVIDUAL MATERNAL OBSERVATIONS
CONTROL
Fluid
Vocal- Nasal	Around Sali-
RAT Ataxia Dyspnea Rales	ization Congestion Nose vation
2012
2013
2014
2016
2019
2024
2026
2028	K	e
2032
2036
2037
2040
2042
2043
2044
2047
2049
2051
2055
2056
Note: Each character indicates the day the condition was observed:
A - GD6, B = GD7, C - GD8.., 1 - PD1, 3 = PD3, 6 - PD6.
A lowercase letter indicates the condition was observed only
within the first 4 hours of dosing on that day.

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APPENDIX 1 (CONTINUED)
2-METHYLHEXANOIC ACID
DEVELOPMENTAL TOXICITY TESTING IN SPRAGUE-DAWLEY RATS
INDIVIDUAL MATERNAL OBSERVATIONS
300 MG/KG
Fluid
Vocal- Nasal	Around Sali-
RAT Ataxia Dyspnea
Rales
ization
Congestion
Nose
vation
2010
BCDEfg

GJ

j
2011 A
BcHIj

AeGj


2015
CDEfJ
C
G


2018
ABEflJ

aDegi
a
a
2021
D

dEfglJ

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APPENDIX 1 (CONTINUED)
2-METHYLHEXANOIC ACID
DEVELOPMENTAL TOXICITY TESTING IN SPRAGUE-DAWLEY RATS
INDIVIDUAL MATERNAL OBSERVATIONS
400 MG/KG
Vocal-
Nasal
Fluid
Around
Sali-
RAT
Ataxia Dyspnea
Rales
ization
Congestion
Nose
vat:
2008
i
AfJ
&
b


2009

CdEfH
C
G
h
h
2017

ABCdGHJ
ag
DEfH
h
h
2020

eGhN

BEj
eh
eh
2025

BCI

Df
a
a
2029
A
AH

BcEGhijN
®j
j
2031

EFGHIJ
abcj
A

e
2035

CGH1JK16

efHN
j
j
2038

GHi
g
N
j
j
2041

a
AB



2046

ADJK

bel
de
ade
2050
i"
EFhljK
E
eg


2052

ABCDEIJ

eFg
ae
ae
2053

CDehj
C

j
j
2054

A
a
E


Other Observations
2050 Red vaginal discharge
2054 Mass: firm, not movable,
right hind leg
CDEFGHIJKN136
Note: Each character indicates the day the condition was observed:
A - GD6, B - GD7, C - GD8... 1 - PD1, 3 - PD3, 6 - PD6.
A lowercase letter indicates the condition was observed only
within the first 4 hours of dosing on that day.

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APPENDIX 2
2-METHYLHEXANOIC ACID
DEVELOPMENTAL TOXICITY TESTING IN SPRAGUE-DAULEY RAT
INDIVIDUAL BODY WEIGHTS (g)
CONTROL
Rat 3
6

Gestation Day
8 10 13
16

20
2012
276
298

308

319

343
383

454
2013
261
279

282

298

327
354

429
2014
261
286

295

305

328
348

408
2016a
249
278

288

294

282
282

283
2019
269
280

293

296

318
351

404
2024
266
279

293

303

325
342

402
2026
253
273

280

290

322
355

425
2028
256
276

274

287

308
333

404
2032
268
283

293

302

312
353

405
2036
258
278

284

296

313
340

394
2037
261
284

293

304

324
350

408
2040
244
271

291

307

310
340

393
2042
252
273

284

296

319
343

412
2043a
242
262

254

265

281
271

278
2044
247
265

276

285

311
348

405
2047a
256
272

274

281

278
279

291
2049
255
277

288

305

323
364

435
2051
255
263

276

299

314
349

408
2055
250
280

285

297

324
348

402
2056
259
285

302

316

350
376

455
MEAN 258.3
S.E. 2.0
N - 17
278.
2.
2
0
288.
2.
1
3
300.
2.
3
2
321.
2.
8 351.
7 3.
6
1
414.
4.
a Not pregnant

-------
APPENDIX 2 (CONTINUED)
2-METHYLHEXANOIG ACID
DEVELOPMENTAL TOXICITY TESTING IN SPRAGUE-DAWLEY RATS
INDIVIDUAL BODY WEIGHTS (g)
300 MG/KG
Gestation Day
Rat
3

6
8

10
13

16

20
2010a
243

253
234

249
266

266

274
2011a
256

281
262

289
297

299

292
2015
257

285
290

278
301

334

389
2018
253

272
250

279
306

315

391
2021
270

296
302

279
309

343

406
2022
261

272
281"

297
323

349

407
2023
250

267
273

287
298

320

398
2027a
245

260
264

267
275

266

270
2030
246

257
237

228
258

286

355
2033
263

287
300

311
331

352

389
2034
251

279
279

288
303

317

374
2039
265

282
294

304
313

335

392
2045a
256

292
300

309
308

308

318
2048
268

284
264

289
306

337

402
2057
272

300
306

326
348

376

432
MEAN 259.
S.E. 2.
N - 11
6
7
280.1
3.8
279.
6.
6
7
287.8
7.5
308.
6.
7
8
333.
7.
1
1
394.
5.
a Not pregnant

-------
APPENDIX 2 (CONTINUED)
2-METHYLHEXANOIC ACID
IMmTT"1? ATniPiTrntT CBAVTi^TTIi/	TVT PTin A/^ttr* IS * T Tl T»t/ n > mn
DEVELOPMENTAL TOXICITY TESTING IN SPRAGUE-DAWLEY RATS
INDIVIDUAL BODY WEIGHTS (g)
400 MG/KG
Gestation Day
Rat
3
6
8
10

13
16
20
2008a
246
258
249
257

274
238
257
2009
274
283
304
302

319
364
422
2017
247
272
250
274

297
304
368
2020
272
295
303
314

326
350
416
2025
253
279
264
295

304
349
406
2029
257
281
243
265

274
292
335
2031
248
269
276
283

292
322
388
2035
255
278
254
288

291
289
362
2038a
242
268
282
286

284
280
274
2041
251
278
273
289

320
345
420
2046
260
291
272
296

323
351
414
2050
265
295
295
309

325
282
343
2052
266
291
255
287

303
331
397
2053
263
292
297
279

322
334
386
2054
256
284
268
288

320
347
411
MEAN 259.0
S.E, 2.4
N - 13
283.
2.
7 273.4
4 5.7
289.
3.
9
.8
308.9
4.6
327.
7.
7 389
6 8
a Not pregnant

-------
APPENDIX 3
2-METHYLHEXANOIC ACID
DEVELOPMENTAL TOXICITY TESTING IN SPRAGUE-DAWLEY RATS
INDIVIDUAL PARTURITION AND POSTNATAL DATA
CONTROL
Day 1	Day 3	Day 6
Gestation Number	Number Number
Length of Pups Weight(g) of Pups of Pups Ueight(g)
Rat Days Rankr Live Dead Litter Pup Live Dead Live Dead Litter Pup Implants
2012
21
3
12
0
88
7
7.4
12
0
12
0
167.7
14.0
12
2013
21
29.5
13
0
81
3
6.3
13
0
12
0
145.6
12.1
13
2014
21
37
8
0
55
0
6.9
8
0
8
0
118.2
14.8
9
2016a



,


.






,
2019
21
29.5
13
1
79
2
6.1
13
0
13
0
163.7
12.6
14
2024
21
10
9
0
67
5
7.5
9
0
9
0
131,3
14.6
13
2026
21
18.5
11
0
90
4
8.2
11
0
11
0
175.1
15.9
12
2028
21
34
12
0
84
6
7.1
12
0
12
0
181.4
15.1
13
2032
22
40
11
0
73
3
6.7
11
0
11
0
146.1
13.3
14
2036
21
3
12
0
89
6
7.5
12
0
12
0
173.5
14.5
14
2037
21
29.5
11
0
78
7
7.2
11
0
11
0
164.0
14.9
11
2040
21
12.5
13
0
100
3
7.7
13
0
13
0
173.7
13.4
14
2042
21
3
13
0
93
1
7.2
13
0
13
0
178.9
13.8
13
2043a



*





,

,

.
2044
21
12.5
11
0
85
9
7.8
11
0
11
0
173.9
15.8
14
2047a

«
,
,










2049
21
3
12
0
94
3
7.9
12
0
12
0
187.3
15.6
13
2051
21
23
14
0
88
8
6.3
14
0
14
0
174.9
12.5
15
2055
21
18.5
11
0
75
6
6.9
11
0
11
0
149.6
13.6
12
2056
21
23
14
0
91
0
6.5
14
0
14
0
184.4
13.2
15
MEAN

19.4
11.8



7.1
11.8

11.7


14.1
13
S.E.

3.1
0.4



0.1
0.4

0.4


0.3
0
N

17
17



17
17

17


17
17
a Not pregnant
r Ranking based on the observed time and stage of parturition.

-------
/
APPENDIX 3 (CONTINUED)
2-METHYLHEXANOIC ACID
DEVELOPMENTAL TOXICITY TESTING IN SPRAGUE-DAWLEY RATS
INDIVIDUAL PARTURITION AND POSTNATAL DATA
300 MG/KG
Day 1	Day 3	Day 6
Gestation Number	Number Number
Length of Pups Weight(g) of Pups of Pups Weight(g)
Rat Days Rankr Live Dead Litter Pup Live Dead Live Dead Litter Pup Implants
2010a
,
,
.
.




.
.


,

2011a
,





,

,


,
,
,
2015
21
15
6
0
50
6
8.4
6
0
6
0
104.1
17.4
11
2018
21
16.5
13
0
84
2
6.5
13
0
13
0
172.3
13.3
14
2021
21
23
11
0
86
7
7.9
11
0
11
0
172.4
15.7
14
2022
21
23
13
0
86
8
6.7
13
0
13
0
181.1
13.9
14
2023
22
39
14
0
95
3
6.8
14
0
14
0
161.3
11.5
14
2027a


.











2030
21
23
14
0
85
3
6.1
14
0
14
0
179.7
12.8
14
2033
21
36
4
0
27
5
6.9
4
0
0
0
,

4
2034
21
8.5
7
0
58
2
8.3
7
0
7
0
132.7
19.0
11
2039
21
3
9
0
69
7
7.7
9
0
9
0
150.6
16.7
10
2045a








,


,
,

2048
21
12.5
9
0
75
8
8.4
9
0
9
0
142.4
15.8
10
2057
21
12.5
9
0
80
4
8.9
9
0
9
0
162.9
18.1
11
MEAN

19.3
9.9



7.5
9.9

9.5


15.4
11.5
S.E,

3.3
1.0



0.3
1.0

1.3


0.8
0.9
N

11
11



11
11

11


10
11
a Not pregnant
r Ranking based on the observed time and stage of parturition,

-------
APPENDIX 3 (CONTINUED)
2-METHYLHEXANOIC ACID
DEVELOPMENTAL TOXICITY TESTING IN SPRAGUE-DAVLEY RATS
INDIVIDUAL PARTURITION AND POSTNATAL DATA
400 MC/KG
Day 1	Day 3	Day 6
Gestation Number	Number Number
Length of Pups Weight(g) of Pups of Pups Weight(g)
Rat
Days Rankr
Live
Dead
Litter
Pup
Live
Dead
Live
Dead
Litter
Pup Implani
2008a













2009
21
6.5
10
0
73,2
7.3
10
0
10
0
144. 3
14.4
14
2017
21
16.5
14
0
84.2
6.0
14
0
14
0
154.4
11.0
14
2020
21
34
11
1
70.7
6.4
11
0

,


13
2025
21
8.5
13
0
98.9
7.6
12
0
12
0
151.4
12.6
14
2029
21
29.5
7
0
51,9
7.4
7
0
7
0
111.0
15.9
13
2031
21
29.5
12
0
80.4
6.7
12
0
12
0
162.0
13.5
14
2035
21
23
11
0
73.2
6.7
11
0
11
0
154.2
14.0
13
2038a
,
,


.
,

»
,




2041
21
34
14
0
97.4
7.0
14
0
14
0
179.5
12.8
15
2046
21
23
11
0
84.7
7.7
11
0
11
0
173.7
15.8
13
2050
22
38
11
0
67.3
6.1
11
0
11
0
123.5
11.2
12
2052
21
6,5
10
0
73.8
7.4
10
0
10
0
132.3
13.2
12
2053
22
41
7
0
50.9
7.3
7
0
7
0
117.9
16.8
7
2054
21
29.5
9
0
63.0
7.0
9
0
9
0
152.2
16.9
10
MEAN

24.6
10.8


7.0
10.7

10.7


14.0
12.6
S.E.

3.3
0.6


0.2
0.6

0.7


0.6
0.6
N

13
13


13
13

12


12
13
a Not pregnant
b Day-6 data excluded from calculations due to malfunctioning water spigot,
r Ranking based on the observed time and stage of parturition.

-------
TICXMtCAL ItlPOftT DATA
1. #«*©** NO. i
EPA/600/1-92/032
JTi PB91-197418
«. rirta Awoiurmn
2-Methylhexanoic Acid Developmental Toxicity Testing
L NVORT OAT*
June 1991
a. ecntoMMiMS OMaANixATio«« cooa
' rt^MOR^otskyi
R.J. Kavlock?
1. XMPOHMiNaOnaANttATION *MO»t NO
t. PIKrOKMINO ORGANIZATION NAM! ANO AOOftlSS
Lantech Env. Tech., Inc. RTP, NC 27709
2USEPA/HERL/PTB (KD-71),RTP, NC 27711
liMftdlAUIklUINThS.
68-02-4450
13. SPONSORING AQtNCV NAMI ANO AOONtU
Health Effects Research Laboratory - RTP, NC
Office of Research and Development
U.S. Environmental Protection Agency
Research Trianale Park. NC 27711
i*. t*#« o* niponr ano 'iinoo covimo
1*. I^OMtODINO AOINev COOI
	EPA/W9/1Q	,	
15 IUl*l»t«M|NTAHY NQTli
il.		
As part of an investigation of the developmental effects and structure-
activity relationships of aliphatic acids, 2-methylhexanoic acid was
administered by gavage to Sprague-Dawley rats on gestation days 6-15 at doses of
0, 300, and 400 mg/kg/day. The dams were allowed to deliver and their litters
were examined through postnatal day 6. Pups that were found dead were examined
for soft-tissue alterations. On day 6, two survivors per litter were preserved
for skeletal examination. Maternal toxicity was demonstrated at both 300 and
400 nig/kg by weight loss and altered respiration (rales, dyspnea). In spite of
the maternal toxicity present, there were no clear toxic effects on development;
litter size, pre- and postnatal viability, and pup weights were unaffected by
treatment. Skeletal examinations of selected pups yielded inconclusive data; a
slight increase in the incidence of lumbar ribs was present a 400 mg/kg, but was
not clearly attributable to treatment.
1J,	KIT WO»0> ANO OOCUMINT
». oue*irro«a
b. lOINfl»lt*l/0*«M INOIO riMMi
c. CO*ati FMtf/Creur
Developmental Toxicity
Structure-Activity Relationship^
Aliphatic Acids
Moalth

iroiinriUTOi; tnmiki	
RELEASE TO PUBLIC
If HCyMiTv cvam 1 i*u
UNCLASSIFIED
Jf, HQ. OP PAOftl
-< cA.
is. ateuNtf v Clam
UNCLASSIFIED
11. Wi41
IP* fmm 181-1 (*~». 4-m	«0
-------