758. Benalaxyl (Pesticide residues in food: 1987 evaluations Part II Toxicology)

BENALAXYL

EXPLANATION

Residues of benalaxyl, a systemic fungicide, were evaluated by
the JMPR in 1986 (Annex 1, FAO/WHO, 1986e). Toxicological data on
benalaxyl is reviewed here.

EVALUATION FOR ACCEPTABLE DAILY INTAKE

BIOLOGICAL DATA

Biochemical Aspects

Absorption, Distribution and Excretion

Studies reviewed by the 1986 JMPR show that benalaxyl is
rapidly metabolized and excreted by rats (Annex I, FAO/WHO, 1986e).

Toxicological Studies

Special Study on Allergic Sensitisation

In the induction phase, groups of 20 female Hartley albino
guinea pigs were injected intradermally (0.1 ml) with Freunds
Complete adjuvent, p-nitroso dimethylaniline (1%) in petrolatum,
mercuric chloride (0.1% in saline) or benalaxyl (5% in paraffin
oil). After 7 days, the respective injection sites were treated
topically for 48 hours with p-nitroso dimethylalanine (25% in
petrolatum), mercuric chloride (1% in petrolatum) and benalaxyl
(25% in petrolatum). The animals were subsequently challenged after
a further 2 weeks with 5% in petrolatum, mercuric chloride (0.1% in
saline) or p-nitroso dimethylnitrosamine (10% in petrolatum) and
results scored 24 hours after removal of the patch. Benalaxyl did
not induce allergic sensitisation while the positive control,
p-nitroso dimethylaniline, gave the expected strong response (Vole
Gera and Vasconi, 1981).

Special Studies on Carcinogenicity

See under long-term studies.

Special Studies on Eye and Skin Irritation

Table 1.

Method Species Route Results Reference

Primary eye Rabbit Intraocular Non-irritant Dal Re & Vole
irritation Gera (1979a)
(Draize Test)

Primary skin Rabbit Dermal intact Non-irritant Dal Re & Vole
irritation Gera (1979b)
(Draize Test)

Special Studies on Mutagenicity

See Table 2.

Special Study on Reproduction

Groups of 25 male and 25 female Sprague-Dawely rats received
benalaxyl (purity unspecified) in the diet at 0, 100, 1,000 and
5,000 ppm for 112 days before intial mating. Similar treatment was
continued throughout two litter and into a second generation which
also produced two litters.

Dietary exposure to benalaxyl had no effect on parental
mortality or on fertility, length of gestation period, litter size
or the number of stillborn pups in either the F1 or F2 generations.
There was a significant reduction in the bodyweight of high dose F1
males and am irregular reduction for high dose F1, F2A and F2B
females. F2B pups of the high-dose group had reduced bodyweights at
irregular intervals.

At necropsy, the high dose F1 females had significantly
increased liver weights whilst F1 rats of both sexes had increased
liver to bodyweight ratios. F2B pups had increased liver to
bodyweight ratios at 1,000 ppm whilst male and female F2B pups
exhibited increased liver to bodyweight ratios at
5,000 ppm. Treatment had no effect on implantation or resorption
rates or on the number of corpora lutea. There were no
histopathological findings that could be related to treatment.
Accordingly, feeding up to 100 ppm benalaxyl in the diet has no
effect on the reproduction of rats (Johnson and Becci, 1983).

Special Studies on Teratogenicity

Rats

Groups of 20-23 gravid female Charles River CD (SD) BR rats
received by oral gavage 0, 12.5, 50 and 200 mg/kg benalaxyl (95%
pure) in methylcellulose (0.5%) from the 6th to 15th day of
gestation. Twenty days after mating, the animals were sacrificed
and their uterine contents examined.

The treatment had no observable effect on the rats and there
was no mortality. There were no significant differences in the
numbers of implantations, post-implantation losses, numbers of
viable fetuses or their sex ratio. No major malformation occurred
in any group but there was a dose-related increase in the frequency
of minor skeletal anomalies, arising especially from delayed
skeletal ossification, which became statistically significant for
the mid and high-dose groups. Accordingly, the results of this
study indicate that benalaxyl is not teratogenic to rats at daily
doses up to 200 mg/kg (Mondino, 1982a).

Rabbits

Groups of 16 mated female New Zealand White Rabbits received,
0, 5, 50 or 250 mg/kg benalaxyl (purity unspecified, in 1% aqueous
methylcellulose) by daily oral gavage from day 6 to 27 of
gestation. The animals were sacrificed on day 28 and their uterine
contents removed and examined.

Seven of the rabbits died during the study, either from
intubation error or respiratory disease but these came from all
groups in similar numbers. High-dose rabbits lost weight during the
last 5 days of the study. At necropsy, the incidences of pregnancy
were similar in all groups. Treatment had no effect on the
implantation rate or post-implantation losses. The mean number of
fetuses and their sex ratios were unaffected but mean litter weight
was slightly reduced in the high-dose group. The mean crown-rump
length was significantly reduced at 250 mg/kg and slightly reduced
at 50 mg/kg. There were no treatment related effects on the rate of
minor skeletal defects. The results of this study indicate that
5 mg/kg daily administered of benalaxyl does not affect the fetal
development of rabbits (Irvine, 1984).

Table 2: Acute Toxicity of Benalaxyl

Species Sex Route Purity LD₅₀/LC₅₀ Reference

Mouse M/F Oral 92.5% 680 mg/kg Dal Re & Vola
Gera, 1981

Rat M/F Oral 96.5% 4,200 mg/kg Dal Re & Vola
Gera, 1979c

Rat M/F Inhalational ? >10 mg/l Sterner &
Pfennig, 1985

Rat M/F Intraperitoneal ? 1,100 mg/kg Dal Re & Vola
Gera, 1979d

Rabbit M/F Dermal 94% 2,000 mg/kg Vola Gera &
Vasconi, 1983

SHORT-TERM STUDIES

Mice

In a dose-ranging study, groups of 20 male and 20 female Swiss
mice received benalaxyl (94% pure) in their diet at 0, 500, 1,000,
2,000, 3,000 and 5,000 ppm for 90 days. There were no apparent
effects on the behaviour, bodyweight, food and water consumption or
animal mortality. Half of the mice of each group and sex were
sacrificed at 40 days for interim evaluation.

At necropsy, there was a dose-related increase in absolute and
relative liver weights which occurred at and above 1,000 ppm at
96 days and at and above 2,000 ppm at 42 days, especially in female
mice. Histopathology showed no significant differences between
treated and control groups (Maltoni, 1985).

Rats

Groups of Charles River CD(SD) BR rats (20 per sex per group)
were fed benalaxyl in the diet at levels of 0, 10, 100, 1000 and
10000 ppm for 13 weeks. A further group (20 per sex) were fed
benalaxyl in the diet at a level of 12000 ppm for 4 weeks followed
by a recovery period of 4 weeks with no chemical in the diet.
Observations included clinical signs, food consumption, body
weight, organ weight, hematology, urinalysis, gross and microscopic
pathology.

There were no treatment related deaths during the study. One
animal in the recovery group died subsequent to blood sampling
which was performed by incision of a sublingual vein under light
ether anaesthesia at 0, 5 and 13 weeks.

There were no clinical signs of treatment. Body-weight gain
was reduced slightly between weeks 2 and 13 at the 10000 ppm level,
and during the treatment period at the 12000 ppm level. Slight
anemia was seen after 5 weeks in females and males at the 12000 ppm
level, but values returned to normal during the recovery period.
Serum cholesterol was increased after 5 and 13 weeks in males and
females at 10000 ppm. In the recovery groups fed 12000 ppm,
cholesterol was elevated at 5 weeks but normal by 13 weeks. Liver
weight was increased at the 1000 and 10000 ppm levels in a dose
related manner.

At necropsy the evidence of "lobulated" liver was increased at
the 10000 ppm level in males. Diffuse hepatic steatosis was
observed in both sexes at this dose level, but was not seen at
sacrifice in the recovery group. There were no other treatment
related histopathological findings. Pituitary adenomas were found
in one female at both the 10 and 100 ppm levels.

The study indicated that feeding of benalaxyl at dietary levels up
to 100 ppm (5 mg/kg b.w./day) is without toxicological effect in
rats (Mondino, 1982b).

Dogs

Groups of 6 male and 6 female Beagle dogs received benalaxyl
(92.5% pure) in the diet at 0, 10, 200 and 800 ppm for 52 weeks.
There were no mortalities and no signs were observed that could be
related to treatment. Ophthalmoscopy, performed at the beginning
and at termination of exposure, was unremarkable. Treatment had no
effect on food consumption or bodyweight gain. Hematological and
clinical chemical parameters and urinalysis, investigated at
regular intervals throughout the exposure, showed no consistent
effects and none that were related to treatment.

At necropsy, there were no remarkable gross pathological
findings. Histopathology showed atrophy of the seminiferous tubules
of two high-dose dogs that was not seen in any other group.
Accordingly, the results of this study indicate that feeding up to
200 ppm in the diet is without observable effect in dogs (Mondino,
1982c).

LONG-TERM STUDIES

Mice

Groups of 60 male and female Swiss mice received benalaxyl
(94% pure) in their diets at 0, 250, 1,000 and 3,000 ppm for 78
weeks. Benealaxyl exposure had no effect on the survival of female
mice but reduced the survival of males treated at 1,000 and 3,000
ppm. The body weight of treated male groups tended to be less than
controls, but there was no association with dietary concentration
of benalaxyl. Treatment had no observable effect on behaviour or
appearance. Clinical chemistry and hematological parameters were
not influenced by treatment.

At necropsy, the absolute and relative liver weights of female
mice exposed to 3,000 ppm benalaxyl was increased. Widespread
amyloidosis, affecting adrenal and salivary glands, kidney, liver,
spleen, stomach, intestine and Zymbal gland, was found more
commonly in treated than control mice, especially mid and high dose
males. There was also an association between the amyloidosis and
benalaxyl exposure in those male mice which died spontaneously
during the study. Three high dose male mice were found to have
transitional cell carcinoma of the urinary bladder which was not
seen in other groups. There were no significant increases in other
tumour types in any group. Due to the increased incidence of
amyloid deposition in all treated groups, this study did not
establish a clear no-effect level (Maltoni, 1985).

Rats

Groups of Charles River CD(SD) BR rats (65 per sex per group)
were fed benalaxyl (purity unstated) in the diet at levels of 0, 4,
100 and 1000 ppm for 104 weeks. A separate group of 30 rats per sex
were used for the 'pre-study' clinical testing programme. Animals
were given fresh diet each week and each batch was analysed for
benalaxyl content.

All animals were observed daily for clinical symptoms of
toxicity. Body weight and food consumption were measured weekly for
14 weeks and twice weekly thereafter. Blood samples for clinical
chemistry and hematology were collected at 0, 3 (hematology only),
6, 12, 18 and 24 months. Urinalysis were performed at 12, 18 and'
24 months; ophthalmological examinations were performed at 0, 1, 6,
12, 18 and 24 months. An interim sacrifice (10 rats/sex/group) was
made at 12 months. All animals except the 'pre-study' clinical
testing group were subject to gross necropsy and histopathological
examination. Organ weights were determined for brain, heart,
kidneys, liver, ovaries, testes and thymus.

No clinical signs of toxicity were apparent during the study.
Mortality was not treatment related; survival at 104 weeks ranged
from 34 to 53%. There were no effects of treatment on body weight
or food consumption. Hematology, clinical chemistry, urinalysis and
ophthalmoscopy were unremarkable. There were no treatment related
effects on organ weights, and there was no gross or microscopic
pathology.

The study demonstrated no toxic effects of treatment of rats
with benalaxyl at levels up to 1000 ppm in the diet (equivalent to
50 mg/kg b.w./day) (Thompson et al., 1983).

Special Studies on Mutagenicity

Table 2 summarizes the results of mutagenicity testing of
benalaxyl.

Table 2. Special Studies on Mutagenicity

Test System Test Object Concentrations Purity Results References

Ames test (with and Salmonella typhimurium 200, 400, 800, 98% Negative de Carneri, 1979a
without metabolic TA1535, TA1537, TA1538, 1600 ug/plate (1)
activation) TA98, TA100

Gene mutation Schizosaccharomyces 20, 40, 80, ? Negative de Carneri, 1980
(without metabolic pombe P1 (Sp 198, 100 ug/ml (2)
activation) (with ade 6-60
metabolic radio-198, h) 100, 200, 400, Negative
activation) 800 ug/ml (3)

Gene mutation Chinese Hamster 3 × 10^-7, 10^-6, ? Negative Monaco, et al.,
(without metabolic V79 Cells 3 × 10^-6, 10^-5, (3) 1983
activation) (with 3 × 10^-5 M
metabolic Negative
activation) (6)

Gene conversion Saccharomyces 8, 40, 200, ? Negative de Carneri, 1979b
(with and without cerevisae D4 1000 ug/ml (2)
metabolic
activation)

Chromosome Chinese Hamster 1000, 2000, ? Negative Mondino, 1980a
Abberation Ovary Cells 4000 mg/kg (5)

Chromosome Cultured human Mondino, 1980b
aberration lymphocytes
(without metabolic 0.083, 0.25, ? Negative
activation) (with 0.83, 2.5 mg/ml (5)
metabolic 0.033, 0.1, Negative
activation) 0.033, 1 mg/ml (7)

Table 2. contd.

Test System Test Object Concentrations Purity Results References

Primary Rat Rat Hepatocytes 0.5, 1.0 94% Negative Myhr and Brusick, 1983
Hepatocyte
Unsheduled DNA 2.5, 5, 10, 25,
Synthesis 50 ug/ml

(1) Positive controls, N-methyl-N-nitro-N-nitrosoquanidine, 2-nitrofluorene and 2-acetylaminofluorine
(2-AAF) gave expected positive result.
(2) Positive control, methyl methanesulphonate, gave expected positive result.
(3) Positive control, N-dimethylnitrosamine, gave expected positive result.
(4) Positive control, cyclophosphamide, gave expected positive result.
(5) Positive control, Mitomycin C, gave expected positive result.
(6) Positive control, ethanemethanesulphonate, gave expected positive result.
(7) Positive control, phenacetin, gave expected positive result.
(8) Positive control, 2-AAF gave expected positive result.

COMMENTS

Benalaxyl was evaluated toxicologically for the first time by
the present meeting. It was evaluated by the 1986 JMPR for residues
only. As noted at that time, benalaxyl is completely metabolized
and does not accumulate in the tissues of rats. Comprehensive
short- and long-term dietary administration indicates that the
toxicity of benalaxyl is low. Hepatic enlargement occurred in rats
fed benalaxyl (> 1000 ppm) in their diet for 14 weeks and in
mice fed for 78 weeks (3000 ppm). However, hepatomegaly did not
occur in rats fed benalaxyl (1000 ppm) for two years or in dogs fed
for one year (800 ppm).

The meeting concluded that although the hepatic enlargement
observed in rodents was of questionable toxicological significance,
it could be used to establish a no-effect level and estimate an
ADI.

TOXICOLOGICAL EVALUATION

LEVEL CAUSING NO TOXICOLOGICAL EFFECT

Mouse: 500 ppm in the diet, equivalent to 75 mg/kg bw/day.
Rat: 100 ppm in the diet, equal to 5 mg/kg bw/day.
Dog: 200 ppm in the diet, equal to 7 mg/kg bw/day.

ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN

0-0.05 mg/kg bw.

STUDIES WHICH WILL PROVIDE INFORMATION VALUABLE IN THE CONTINUED
EVALUATION OF THE COMPOUND

Observations in man.

REFERENCES

de Carneri, I., 1979a. Microbiological Mutagenesis Study on CRA 109
(M9834). Genetic mutation test in Salmonella typhimurium (Ames).
Farmitalia Carlo Erba Research and Development. Unpublished study
submitted to WHO by Farmoplant Research and Development.

de Carneri, I., 1979b. Microbiological Mutagenesis Study on CRA 109
(M9834). DNA damage and repair test (Mitotic gene conversion in
Saccharomyces cerevisae D4). Farmitalia Carlo Erba Research and
Development. Unpublished study submitted to WHO by Farmoplant
Research and Development.

de Carneri, I., 1980. Microbiological Mutagenesis Study on CRA 109
(M9834). In vitro gene mutation test in Schizosaccharomyces pombe
P1. Farmitalia Carlo Erba Research and Development. Unpublished
study submitted to WHO by Farmoplant Research and Development.

Dal Re, U. & Vola Gera, F., 1979a. Primary eye irritation study of
M9834 in albino rabbits. Montedison Division Agricoltura. Centro
Ricerche Antiparassitari. Unpublished study submitted to WHO by
Farmoplant Research and Development.

Dal Re, U. & Vola Gera, F., 1979b. Primary skin irritation study of
M9834 in albino rabbits. Montedison Division Agricoltura. Centro
Ricerche Antiparassitari. Unpublished study submitted to WHO by
Farmoplant Research and Development.

Dal Re, U. & Vola Gera, F., 1979c. Acute oral toxicity study of
M9834 in albino rat. Montedison Division Agricoltura. Centro
Ricerche Antiparassitari. Unpublished study s Submitted to WHO by
Farmoplant Research and Development.

Dal Re, U. & Vola Gera, F., 1979d. Determination of the acute
intraperitoneal toxicity of M9834, technical grade, in the rat.
Montedison S.p.A. Agricultural Division, Pesticide Research Centre,
Milan. Unpublished study submitted to WHO by Farmoplant Research
and Development.

Dal Re, U. & Vola Gera, F., 1981. Determination of the acute oral
toxicity of Galben, technical grade, in albino mice. Gruppo
Montedison S.p.A. Pesticide Research Centre, Milan. Unpublished
study submitted to WHO by Farmoplant Research and Development.

Irvine, L.F.H., 1984. Galben: Oral (Gavage) teratology Study in the
New Zealand White Rabbit. Hazleton Laboratories Europe Ltd Report
No. 3596-309/3. Unpublished study submitted to WHO by Farmoplant
Research and Development.

Johnson, W.D. & Becci, P.J., 1983. Two generation Reproduction
Study in Rats with Galben Technical (Final Report). Food and Drug
Research Laboratories, Inc. Waverly Study No. 7220. Unpublished
study submitted to WHO by Farmoplant Research and Development.

Maltoni, C., 1985. Evaluation of Chronic Toxicity and Oncogenic
Potential of Galben (R) (CAS no. 71626-11-4) in Swiss Mice (Oral
Dosing Study). Bologna Institute of Oncology Study No. BT 5004.
Unpublished study submitted to WHO by Farmoplant Research and
Development.

Monaco, M., Forster, R. & Nunziata, A., 1983. Gene mutation in
Chinese Hamster V798 Cells Test substance: Galben TH. Life Sciences
Research, Roma toxicology Centre Rep. No. LSR-RTC FMP 001.
Unpublished study submitted to WHO by Farmoplant Research and
Development.

Mondino, A., 1980a. In vivo Study of the Induction of Chromosome
Aberrations in the Chinese Hamster by Compound M 9834 Administered
Orally. Institute of Biomedical Research Antoine Marxer S.p.A.
Ivrea. Unpublished study submitted to WHO by Farmoplant Research
and Development.

Mondino, A., 1980b. In vitro Study of the Induction of Chromosome
Aberrations by Compound M 9834 in Human Lymphocyte Cultures.
Institute of Biomedical Research Antoine Marxer S.p.A. Ivrea.
Unpublished study submitted to WHO by Farmoplant Research and
Development.

Mondino, A., 1982a. Teratogenesis study of the product M 9834 in
Charles River CD (SD) rats on oral administration from the 6th to
24th day of gestation, at daily doses of 12.5, 50 and 200 mg/kg.
Antoine Marxer Institute of Biomedical Research, Ivrea. Unpublished
study submitted to WHO by Farmoplant Research and Development.

Mondino, A., 1982b. 13-Week Oral Subacute Toxicity study of the
Product M 9834 (Galben) administered to Charles River CD (SD) BR
rats in the diet, at the doses of 10, 100, 1000, 10,000 and
12,000 ppm. Institute of Biomedical Research 'Antoine Marxer',
S.p.A. Ivrea. Unpublished study submitted to WHO by Farmoplant
Research and Development.

Mondino, A., 1982c. Long term toxicity study with repeated oral
administration of the product M 9834 (Galben) added to the food of
Beagle dogs for 52 weeks, at the dosage levels of 10, 200 and
800 ppm. Antoine Marxer Institute of Biomedical Research, Ivrea.
Unpublished study submitted to WHO by Farmoplant Research and
Development.

Myhr, B.C. & Brusick, D.J., 1983. Evaluation of Galben in the
Primary Rat Hepatocyte Unscheduled DNA Synthesis Assay. Final
Report, Litton Bionetics Project No. 20991. Unpublished study
submitted to WHO by Farmoplant Research and Development.

Sterner & Pfennig, 1985. Acute Inhalation Toxicity of 'Galben Th'
in Rats. IBR Forschungs GmBh, Walsrode Proj. No. 1-4-549-81.
Unpublished study submitted to WHO by Farmoplant Research and
Development.

Thompson, S.W., Davidson, T.J. & Becci, P.J., 1983. Lifetime Oral
Dosing Studies in Rats: Combined Oncogenicity and Chronic Toxicity
of Galben Technical 9834. Food and Research Laboratories, Inc.
Report No. 6568-11 (Final Report). Unpublished study submitted to
WHO by Farmoplant Research and Development.

Vola Gera, F. & Vasconi, F., 1981. Allergic contact sensitisation
test on albino guinea pig using the maximisation test: evaluation
of the allergenic potency of Galben. Farmoplant Centro Ricerche
Antiparassitari Biological Evaluation Department, Milan.
Unpublished study submitted to WHO by Farmoplant Research and
Development.

Vola Gera, F. & Vasconi, F., 1983. Galben Acute Dermal Toxicity
Study (LD₅₀) in the Albino Rabbit. Limit Test. Farmoplant Centro
Ricerche Antiparassitari Biological Evaluation Department, Milan.
Unpublished study submitted to WHO by Farmoplant Research and
Development.

    See Also:
       Toxicological Abbreviations
       Benalaxyl (JMPR Evaluations 2005 Part II Toxicological)