Evaluation of the Toxicity and Teratogenicity of Benzene Amino and Nitroderivatives for their Regula

1
Evaluation of the Toxicity and Teratogenicity of
Benzene Amino- and Nitroderivatives for their
Regulation in Occupational Air.K. Kabirov, A.
Lyubimov,Toxicology Research Laboratory,
University of Illinois at Chicago, 1940 W. Taylor
St., Chicago, IL 60612
Cliff Avoidance
NE
NE
NE
Surface Righting Reflex
NE
NE
NE
Skin Pain Threshold
NE
NE
NE
Abstract ID946
ABSTRACT Amino- and nitroderivatives of benzene
are widely used in the chemical industry as
intermediate products in the manufacturing of
dyes, medicines, pesticides, etc. In order to
provide safety to people who have contact with
the amino- and nitroderivatives of benzene, it is
necessary to study not only the acute and chronic
toxicity of these substances, but also the
long-term effects such as embryotoxic effect.
Our own studies, and studies of other authors,
showed that under conditions of inhalation
exposure for 4 hr/day, 7 days/week, throughout
gestation of the Wistar rat, such compounds as
2-nitro-4-bromophenol, the 3-nitrobenzoic acid,
3,4-difluoronitrobenzene, 3,4-difluoroaniline,
ortho-phenylenediamine, 2,4- dinitroaniline,
nitrobenzene, 2,4-dichloronitrobenzene expressed
an embryotoxic effect. This effect includes
increased embryo losses, teratogenicity, changes
in the central nervous system and the
cardiovascular system, and delays in the physical
development of progeny. The Lowest Observed
Adverse Effect Levels (LOAELs) for embryotoxic
effect were established at lower levels than the
LOAELs for maternal toxicity in pregnant dams.
On the basis of Quantitative Structure-Activity
Relationship (QSAR) analysis, the equations for
prediction of both the LOAELs for maternal
toxicity and for embryotoxic effects of amino-
and nitroderivatives of benzene were obtained.
This gives a possibility of approximate
determination of the toxic effect of amino- and
nitroderivatives of benzene (including
embryotoxicity) at the stage of compound rational
design (even before its synthesis). Thus, in our
opinion, the occupational amino- and
nitroderivatives of benzene may be potent
embryotoxic agents, which is necessary to
consider during the evaluation of permissible
levels of exposure.
Table 1. Summary of Maternal Toxicitya

Table 3. Summary of Offspring Physical
Developmenta
Figure 2. Ear Unfolding and Hair Appearance of
Offspring from Females Inhaling DFNB Throughout
the Entire Pregnancy

Introduction Amino- and nitroderivatives of
benzene, such as 2-Nitro-4-bromophenol (NBP),
3,4-Difluoroaniline (DFA), 3,4-Difluoronitrobenzen
e (DFNB), and 3-Nitrobenzoic Acid (NBA), are
widely used in the manufacturing of
pharmaceuticals, dyes, pesticides, plastics and
other products. Many women have contact with
these compounds in the chemical industry.
Therefore, NBP, DFA, DFNB and NBA were studied
for maternal and postnatal toxicity,
embryotoxicity and teratogenicity in rats
inhaling these chemicals 4-hr/day during the
entire period of pregnancy. The objective of
this study was to determine the developmental
toxicity of NBP, DFA, DFNB and NBA in Wistar
rats. Experimental Design Test Articles NBP,
DFA, DFNB and NBA, research grade, from the local
manufacturer (NPO The State Institute of Applied
Chemistry, Perm, Russia). The structures of the
chemicals were confirmed by mass spectroscopy.
Animals Wistar rats obtained from the
Rappolovo animal facility, St. Petersburg,
Russia, were 10-12 weeks old upon arrival.
Pregnant dams weighed 180-200 g at inhalation
initiation. Animals for
natural delivery Animals were randomized by
weight into each group of 20 animals/group. Statis
tical analysis For analysis of continuous
outcome variables, one-way ANOVA was used.
Dunnet's two-tailed t-test was used to identify
the concentrations differing from control values.
Differences were considered statistically
significant at plt0.05. For analysis of prenatal
and postnatal mortality, and incidence of
visceral and skeletal variants, the Wilcoxon
rank-sum test was used. Each concentration was
compared to the control, and Boferroni adjustment
was used.
Table 2. Summary of Embryotoxicity and
Fetotoxicitya
Table 4. Summary of Offspring Neurobehavioral
Developmenta
Table 6. Equation for Prediction of the LOAELs
of Subchronic Inhalation Toxicity (by whole body
inhalation for 4 hr/day daily/4 months) and
Embryotoxic Inhalation Toxicity (by whole body
inhalation for 4 hr/day, 7 days/week throughout
gestation) of Amino- and Nitrobenzenes.
Aminobenzenes log LOAEL SCH -0.269 0.464
log P
r 0.805 log LOAEL SCH -6.226 2.544 R

r 0.677 log LOAEL SCH
5.485 0.305 log P 7.024 Q MAX - 15.842 E N
r 0.812 Nitrobenzenes log LOAEL
SCH -15.358 34.739 N MAX r
0.875 log LOAEL SCH -17.108 32.426 N MAX
0.516 R r 0.881 Amino- and
Nitrobenzenes log LOAEL SCH -0.146 0.432
log P r 0.702 Amino- and
Nitrobenzenes log LOAEL EMBR 6.045 - 13.862 N
MAX r 0.867 log LOAEL EMBR
0.427 - 10.380 Q MAX r 0.812
where log P Hanch lipophylic constant
(Hanch C., Leo A., Unger C., et. al., 1973) R
the energy of resonance E N the connection
energy of amino- of nitro group with the benzene
ring N MAX the index of free valence Q MAX
the maximum charge of a carbon atom not bound to
the substitute. The values of R, E N, N MAX and
Q MAX were calculated by using the method of
molecular orbitals as a linear combination of
atomic orbits (MO LCAO) in the Hukkel
p-electronic approximation (Dewar M., 1972
Dyachkov P.N., 1990).
Table 5. Summary of Offspring Necropsy and
Hematologya
Figure 1. Total Embryolethality in Female
Inhaling NBF, DFA, DFNB and NBA Throughout the
Entire Pregnancy
Measurements Maternal toxicity Body weights and
rectal temperature were recorded at randomization
(Gestation day 0 or GD0) and on GD20 just prior
sacrifice. CNS Skin pain threshold (SPT),
Behavior reactions in an open field. Hematology
Hemoglobin, Methemoglobin, Erythrocyte count,
Leukocyte count and differential. Clinical
chemistry Alanine aminotransferase (ALT),
Aspartate aminotransferase (AST), Cholinesterase,
Catalase, Peroxidase, Alkaline phosphatase, Blood
urea nitrogen, and Urea. Urinalysis Total amino
nitrogen, Urea, Chloride in urine, Phenols
excretion, 24-hr urine volume, Specific gravity.
Liver damage Lipid peroxidation, Relative liver
weights. Hormone metabolism changes
Progesterone, Estradiol and Corticosterone in
plasma. Necropsy 12 animals/group were
necropsied on GD20. Embryotoxicity and
fetotoxicity Total embryolethality,
Pre-implantation loss, Post-implantation loss,
Weights of fetuses, Lengths of fetuses, Weights
of placentas, External and Visceral abnormalities
(Wilson, 1965), Skeletal defects (Dawson, 1926).
Postnatal developmental and neurobehavioral
effects Pup death on 5th and 21st day of
gestation, Ear unfolding, Hair appearance, Upper
and lower incisors eruption, Eye opening,
Offspring weights, Offspring lengths, Behavior
reactions in an open field, Cliff avoidance,
Surface righting reflex, SPT, Relative organ
weights at necropsy, Erythrocyte count, Leukocyte
count and differential.
SUMMARY 1. The maternal toxicity of the pregnant
dams exposed to the studied amino- and
nitroderivatives of benzene was mainly
characterized by damage to liver and kidney
functions. 2. In pregnant dams, the
Lowest-Observed-Adverse-Effect-Levels (LOAELs)
for NBF, DFA, DFNB and NBA were 2.6, 0.9, 0.9 and
1.0 mg/m3, respectively. 3. The embryotoxicity of
studied compounds was displayed not only by
embryolethality, but also teratogenic and
fetotoxic effects. 4. The LOAELs for
embryotoxic activity of NBF, DFA, DFNB and NBA
are 2.6, 0.9, 0.9 and 1.0 mg/m3, respectively. 5.
The LOAELs for embryotoxic activity of NBF, DFA,
DFNB and NBA were 2.5-3 fold lower, than their
corresponding LOAELs for maternal toxicity. 6.
The suggested computational models allow
prediction of embryotoxicity of amino- and
nitroderivatives of benzene prior to their
laboratory synthesis for selection of the least
toxic substances.
Table 5. Lowest Observed Adverse Effect Levels
(LOAELs) for Maternal Toxicity and
Embryotoxicity