Antigestational effects of Icon®, a pyrethroid insecticide on mid pregnancy of rats

Icon 9 is a water miscible type II synthetic pyrethroid insecticide based on active ingredient lambda cyhalothrin (10% w/v). It was recently introduced to Sri Lanka as an indoor spray against malaria vector mosquitoes. The aim of this study was to ascertain the potential effects of Icon 9 on pregnancy outcome of rats when exposed during mid pregnancy (days 8 -14). Icon 9 was orally administered daily during this period in three different doses: 63, 83, 125 mg/kg body wt./day of Icon 9 (active ingredient; lambda cyhalothrin ; 6.3, 8.3, 12.5 mg/kg/day) respectively. Several parameters of reproduction, pre- and post-natal development were monitored. The results demonstrated that Icon® is detrimental to pregnancy outcome (in terms of number of uterine implants, number of viable implants, post implantation loss, number of pups born, litter index, and foetal survival ratio) but induced no changes in gestational length, gross morphological birth abnormalities or detectable pre- and post- natal developmental impairments. The anti-reproductive effects of Icon 9 were mediated by multiple mechanisms (hypophagia, maternal toxicity, stress, uterine myotropic activity, embryo-foetoxicity, anti-progestrogenic activity, inhibition of decidualizaton process, abortifacient activity and vaginal bleeding) leading to enhancement of post-implantation losses. It is concluded that exposure to Icon 9 during mid gestation poses a considerable threat to pregnancy of rats.


Introduction
Icon 9 , produced by the Public Health Division of the Imperial Chemical Industries in England (currently known as Zeneca) (1) is a water miscible type II synthetic pyrethroid insecticide having lambdacyhalothrin (10% w/v) as the active ingredient (2). Information on inert fillers, adjuvants, excipient, wetting agents and purity are not available. This insecticide was recently introduced to Sri Lanka to use as an indoor spray against malaria vector mosquitoes. Icon® is a contact and residual insecticide and acts as a neuropoison by interfering with the conductance of nerve membrane by prolonging the sodium current (2,3). Pyrethroids are also known to increase spontaneous release of neurotransmitters such as GABA, dopamine and noradrenaline, and also act as a hormone disruptor (4, 5).
The effective spray dose of Icon® in Sri Lanka is 312 mg/m 2 (CIC brochure and personal communication K. Gunasekara, Parasitologist, Anti Malaria Campaign, Sri Lanka,). Insecticide residues in indoor environments are more persistent than in the environment at large, as indoors residues are not subjected to degradation by sun, rain and soil microbes and hence there is a potential risk that inhabitants in Icon®sprayed houses are continually subjected to dietary, respiratory and cutaneuos exposures (2).
Recently we showed that Icon® exposure to male rats impaired sexual competence (6) and in female rats when exposed during early gestation was detrimental to their pregnancy outcome (7). Several pesticides when exposed during mid pregnancy are known to interrupt pregnancy (8, 9, 10, 11, and 12). However, the potential antireproductive effects of Icon® when exposed during mid pregnancy is not known and this is worth examining as Icon 91 is an endocrine disruptor (7).
The aim of this study was to investigate the effects of Icon® on reproductive outcome of female rats subjected to repeated exposure during mid pregnancy. This was done using doses of Icon® (approximately 2.5 to 4 times lower than the recommended spray dose in Sri Lanka) identical to what has been used to ascertain reproductive effects when exposed during early pregnancy (7).

Animals
Healthy adult cross bred female rats (weight: 200-250 g) and male rats (weight: 200-250 g) of proven fertility from the colony maintained at the Department of Zoology, University of Colombo were used. They were maintained singly in plastic cages under standardized animal house conditions (temperature: 28-30 °C; photoperiod: approximately 12 h light/12 h dark; and relative humidity: 50-55 %) with free access to pelleted food (Master Feeds Lanka Ltd., Colombo, Sri Lanka) and tap water. Except at the time of experiment the animals were handled only during cage cleaning.

Icon® preparation
Icon® was obtained from Anti-Malaria Campaign, Narahenpitiya, Colombo 5. Sri Lanka. Three doses [63 (low), 83 (mid) and 125 (high) mg/kg/day; (containing active ingredient lambda cyhalothrin 6.3, 8.3, 12.5 mg /kg/day respectively)] of Icon® in 1ml aliquots were prepared by mixing Icon® in distilled water (DW). These doses are comparable to what has been used previously by us to investigate the potential reproductive effects on male rats (6) and identical to what has been used to test the anti-reproductive effects on female rats when exposed during early pregnancy (7).

Icon® administration
Icon® or vehicle (DW) was orally administered by gastric intubation (09.00 -10.00 h) from days 8-14 of pregnancy. The rats were made pregnant by individually pairing pro-oestrous females overnight with a male rat. Vaginal smears were examined on the following morning (08.00-09.00h) for presence of spermatozoa (considered as day 1 of pregnancy).

Adverse effects
Following every dosing, cage side observations were made on each rat continuously for 3-5 h for mortality,, overt signs of toxicity (salivation, lacrymation, ptosis, squinted eyes, wilting, tremors, Convulsions, ataxia, yellowing and loss of fur), stress (exophthalmia, erection, of fur), lethargy (reduction of spontaneous walking movements, climbing in cage, cleaning of. fur) recumbence, aversive behaviours (self biting and scratching, licking of tail and/or paw, intense self grooming behaviour and vocalization), diarrhoea, colour and odour of urine and vaginal bleeding. In addition, the rectal temperature was recorded on day 8 of pregnancy before dosing and on day 14 of pregnancy (5 h after dosing) using a clinical thermometer (Oson Duopris Company Ltd., Berlin, Germany).

Effect on food and water intake
Food and water intake of Icon 9 treated rats [low (n = 12), mid (n = 12), high (n = 9)] or control (n =12) were determined daily from days 8-14 of treatment using conventional laboratory techniques (13).

Evaluation of sedative potential
The sedative potential of Icon 9 was evaluated using the rat hole-board technique (15). Forty-five, day 8 pregnant rats were assigned into 4 groups and orally treated with Icon 9 [low (n = 12), mid (n = 12), high (n = 9)] or vehicle (n =12) for 7 consecutive days. Five hours after the last dosing, rats were individually placed on the centre of the rat hole-board and were given 7.5 min trial periods. During this period the number of head dips, rears and locomotary activity were noted.

Effect on righting reflex
The time for righting reflex on rats used in evaluating the sedative potential were determined 2 h prior to treatment and immediately after hole-board test as described by Mortin et. al., (16).

Effect on pregnancy outcome and preand post-natal development of pups
Forty-five, day 8 pregnant rats were assigned into 4 groups and orally administered with either different doses of Icon 9 [low (n = 12), mid (n = 12), high (n = 12)] or vehicle (n = 12) for 7 consecutive days.
On day 17 of pregnancy, these rats were subjected to a laporatomy under mild ether anesthesia with aseptic precautions. Upon laporatomy, the total number of uterine implants, the number of viable implants (with pulsating blood vessels and reddish appearance), and the number of dead implants (with non pulsating blood vessels and bluish appearance) were recorded. The appearance and number of corpora lutea in ovaries were also noted. Furthermore, the distribution of embryos in the uterine horns, and the diameter of the first embryo at the ovarian end were measured using a vernier caliper (Gallenkamp, Loughborough, UK). Subsequently the laparatomy incision was sutured. Tetracycline ointment (Wockhardt Veterinary Ltd., Bombay, India) was applied and was injected with a subcutaneous dose of 0.1 ml tetracycline (Wockhardt Veterinary Ltd., Bombay, India). Then the animals were allowed to recover and deliver. The gestation length was recorded.
After delivery, on postnatal day 1, the pups were closely observed and the total number of pups, the total number of viable pups, their body weights (using an electronic balance), cranial length, cranial diameter, cranio-sacral length and tail length of each pup (using a vernier caliper) were determined. The presence of gross external congenital abnormalities were also noted (amelia, anomaly of tail, clubfoot, oligodactyly or syndactyly). Then the pups were observed once daily for the appearance of fur and opening of eyes. The following reproductive indices were computed using the reproductive findings.

Statistical analysis
Data are expressed as means ± standard error of mean (SEM). Mann-Whitney Latest and G-test were used as appropriate. P < 0.05 was considered as statistically significant.

Adverse effects
No deaths were recorded in any of the treated rats. Further, none of the treated rats showed, lacrymation, ptosis, squinted eyes, wilting, tremors, convulsions, yellowing and marked loss of fur, and aversive behaviours. Table 1, Icon® produced ataxia, exophthalmia, piloerection, salivation (low dose: around facial region; mid dose: neck region; and high dose: almost throughout the entire body), vaginal bleeding (between days 9-12 of pregnancy), diarrohea (between days 9-14 of pregnancy} and pale yellow (low dose) to dark yellow (mid dose and high dose) coloured urine with an odour similar to that of Icon® between days 8-14 of pregnancy. These toxic effects were evident from 30-60 min. of administration of Icon® and were completely abolished on the following morning except with the high dose where the effects lasted for 2 days after the full treatment course. Icon® administration did not significantly alter the rectal temperature of any treated rats (P > 0.05; Mann-Whitney i/-test).

Reproductive out come, pre-and post natal development of pups
The results obtained are summarized in Tables 2 and 3. Ail treated rats had normal numbers of reddish, rounded, seemingly healthy looking corpora lutea. The low dose of Icon 9 when administered between days 8 -14 of pregnancy had no significant effect on any of the variables investigated. In contrast, the mid dose significantly impaired the number of uterine implants (by 28%), the number of viable implants (by 37%), the number of pups born (by 37%), the number of live pups born (by 40%), the litter index (by 44%), and the foetal survival ratio (by 34%). Further, these anti-reproductive effects were accompanied by a marked and significant increase of post-implantation losses (mid dose by 82% and high dose by 1512%). In contrast, with the high dose of Icon 9 , no uterine implants were seen with a 100% post-implantation loss. Because of this the other variables listed could not be investigated.
The pups born with both low and mid doses of Icon 9 appeared normal with no gross external abnormalities. None of the pre-and post-natal developmental variables investigated were significantly altered both by the low and mid doses of Icon 9 (Table 3).

Discussion
This study examined the anti-gestational effects of Icon 9 when exposed orally during mid pregnancy of rats (days 8 -14). The results demonstrated that, in rats, repeated oral exposure of Icon 9 during mid gestation is detrimental to pregnancy outcome (in terms of the number of uterine implants, number of viable implants, post implantation loss, number of pups born, litter index and foetal survival ratio). Previous experiments have shown that exposure of rats to Icon' 1 ' during early pregnancy does not produce any impairment in pre-and post-natal development of pups or gross birth defects (7). Still we cannot completely rule out any 7r0w®-induced minor and subtle effects on embryo/ feototoxicity and pup development. In contrast, both teratogenic and developmental retardation have been reported with some pyrethroid (9, 17, 18), carbamate (10, 11), organophosphate (12) and organochlorine (19) insecticides following mid pregnancy exposure. (20,21). This seems to be the case with the current study as well. The main mechanism of Icon 9induced disruption of mid pregnancy is due to a marked potentiation of postimplantation loss. On the other hand, Icon® exposure during early gestation is known to interrupt pregnancy both by increased preand post-implantation losses (7). In the rat, uterine implantation occurs on day 4 of pregnancy (22). Hence, pre-implantation loss cannot account for any of the antireproductive effects seen in this study.

It is generally accepted that pregnancy disruption by reproductive toxicants are mediated by different mechanisms
Non-specific and specific mechanisms can potentiate post-implantation loss. Impairment of food intake and maternal weight loss (23) are two of the non-specific factors. In this study, Icon 91 induced maternal weight loss is likely to contribute to the observed anti-gestational effects. Several maternal toxicants interrupt pregnancy (20, 21) and this mode of action is also likely to be operative here as marked toxic signs were evident (ataxia, salivation, exophthalmia, piloerection, depressed body weights, food consumption, production of dark yellow coloured urine and diarrohea)). Stress is another non-specific mechanism that can induce post-implantation losses (24). In rats, stress inhibits luteal function during mid pregnancy causing foetal resorption (25). In this study the Icon 9 treated rats showed features of stress such as piloerection, exopthalmia and this may have contributed to foetal resorption. Other investigators have also shown pyrethroid insecticides like cypermethrine (26) and carbamate insecticides like carbofuran (8) to be producing stress, particularly in mid pregnancy period. Further, we have previously shown increased adrenal weights and enlargement of zona fasciculata of adrenal cortex following Icon 9 treatment (7). Thus, it is likely that stress has played an important role in /row®-induced pregnancy impairment which was seen in this study.

Sedatives are known to interrupt pregnancy (27). Such a mode of action is unlikely in this study as none of the variables of the rathole board technique were inhibited: a reliable and a sensitive test used widely to evaluate potential sedatives (15).
A specific mechanism that could increase post-implantation losses in this study is by a direct embryo/foetocidal action of Icon 9 . A marked loss of viability of foetuses (as judged by lack of pulsations in vitelline vessels and presence of dark blue uterine implants at laporatomy) with mid dose and a total lack of foetuses was seen with the high dose. Further, Icon 9 has been shown to kill both human spermatozoa and Anemia nauplii in vitro (7), which provide additional support to the above mechanism. Further, maternal toxic agents consumed during pregnancy are claimed to be embryo/ foetocidal (24) and also several insecticides are known to posses embryo/foetocide activities (8, 10, 12).
In this study, both mid and the high doses of Icon 9 induced moderate to severe vaginal bleeding which was not accompanied with preterm delivery or pups born small for gestational age. However, shortening (28) and prolongation (8, 29) of gestation durations are reported with some pesticides. In contrast, no vaginal bleeding had been reported during early pregnancy of rats following Icon 9 exposure (7) but it did not result in a pre-term delivery or pups born small for gestational age.
Death of foetus in this study may also results from prolonged uterine contractions due to direct myotropic action of Icon 9 on uterine smooth muscle (7) or by an impairment of progesterone release and or progesterone antagonistic activity (30). Indeed, we have previously shown that Icon 9 induced both tonic and phasic contractions in isolated rat uterine preparations (7) and that it has antiprogestrogenic actions (7). An impairment of progesterone release may result due to stress (25) or due to negative effects of hypophagia and weight loss on the generation of GnRH pulses (31).
Foetal deaths could occur with Icon 9 by these mechanisms also. Progesterone/oestradiol ratio plays a crucial role in maintaining viability of embryos (32). Although Icon 9 is not oestrogenic it is anti-progestrogenic (7) and this effect could alter the ratio and make the uterine environment hostile to embryos, leading to their deaths. This study, together with our previous investigations (7) shows that repeated oral exposure of Icon 9 during both early and mid gestation is detrimental to pregnancy outcome in rats, but induced no detectable gross developmental defects. Icon 9 being an indoor spray, the pregnant domestic animals are liable to get affected..