Characterization of Maternal Influence on Teratogenicity: An Assessment of Developmental Toxicity Studies for the Herbicide Cyanazine1,2

Regulatory Toxicology and Pharmacology 29, 88 –95 (1999) Article ID rtph.1998.1276, available online at http://www.idealibrary.com on

Characterization of Maternal Influence on Teratogenicity: An Assessment of Developmental Toxicity Studies for the Herbicide Cyanazine 1,2 Poorni Iyer, 3 Derek Gammon, Joyce Gee, and Keith Pfeifer Medical Toxicology Branch, Department of Pesticide Regulation (DPR), California Environmental Protection Agency (Cal-EPA), Sacramento, California 95814 Received October 16, 1998

toxicity of a chemical. Accordingly, a systematic and critical approach is needed to characterize the role of maternal toxicity in laboratory animal studies with adverse developmental outcome. The data from developmental toxicity studies for cyanazine have been evaluated employing such an approach. Cyanazine belongs to the triazine family of herbicides and is used worldwide for early preplant, preemergence, or postemergence weed control in corn, cotton, grain, sorghum, and fallow cropland. The structure of cyanazine is presented in Fig. 1. Studies to determine the developmental toxicity of pesticides are conducted according to the guidelines of FIFRA, the Federal Insecticide Fungicide and Rodenticide Act Section 1366 (USEPA, 1982), and are submitted to regulatory agencies. To evaluate the developmental toxicity of cyanazine, the four studies reviewed by Cal-EPA/ DPR included three in the rat and one in the rabbit (Lochry, 1985; Lu et al., 1981; Lu, 1983; Dix et al., 1982).

The contribution of maternal toxicity to the teratogenic effects of the herbicide cyanazine has been assessed to determine whether it may be a hazard to development. Eye defects such as anophthalmia and microphthalmia were observed in rat fetuses and pups. Maternal toxicity was determined from body weight data and clinical signs. Two approaches were used. First, the timing of maternal toxicity was correlated with the specific period of gestation during which the observed fetal defect was most likely to have occurred. Second, individual dams, as well as mean values for each group, were evaluated. The data at the individual level, i.e., in dams with affected litters, did not support conclusions based on the group means. Instead, it is suggested that the developmental effects were not a direct result of maternal toxicity of cyanazine. Data from a rabbit developmental toxicity study supported the findings from the Fischer 344 rat studies. The strategy employed may thus enable direct toxicity to the fetus to be distinguished from developmental toxicity arising as a secondary consequence of maternal toxicity.

RESULTS

Rat Teratology INTRODUCTION

Technical cyanazine was administered daily to groups of 70 mated rats (Fisher 344) per dose by gavage at 0, 5, 25, or 75 mg/kg/day during gestation days 6 –15 (Lochry, 1985). One-half of the dams underwent Caesarian section (C-section) and were killed on gestation day 20; the other half were allowed to deliver naturally. Fetuses were examined on gestation day 20 and pups were examined after natural delivery. In dams, decreased weight gain and food consumption were noted at all dosage levels. At 25 and 75 mg/kg/ day, an increase in clinical signs (chromodacryorrhea, lacrimation, chromorhinorrhea, excess salivation, and soft or liquid feces) was observed. Ataxia, tip-toe walk, thin dehydrated appearance, hyperpnea, inflamed perineum, alopecia, and ptosis were also observed at 75 mg/kg/day. The high dose produced gastrointestinal and liver lesions and was lethal to 13/70 (19%) of the

The importance of maternal toxicity in contributing to the teratogenicity of chemicals has received considerable attention (Kimmel and Wilson, 1973; Palmer and Kavlock, 1987; Schardein, 1987; Khera, 1987), particularly in deciding whether or not a chemical shows selective embryopathy. The relationship between maternal and developmental toxicity is important in making regulatory decisions regarding the developmental 1 The views expressed are the authors’ and do not necessarily represent official policy of California EPA/DPR. 2 Presented in part at The International Congress of Toxicology (Iyer et al., 1995). 3 To whom correspondence should be addressed at Medical Toxicology Branch, Department of Pesticide Regulation, California Environmental Protection Agency, 830 K St., Sacramento, CA 958143510.

88 0273-2300/99

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at 25 and 75 mg/kg/day. The number of resorptions was increased significantly (P , 0.01) at the highest dose. In addition, live litter size, body weight, and survival to day 21 of lactation were decreased. Accordingly, the NOEL for developmental toxicity was 5 mg/kg/day. The effects of cyanazine on various reproductive and fetal parameters are summarized in Table 1. FIG. 1. Structure of cyanazine.

Characterization of Developmental Toxicity

dams, usually after two or three dosings. Hence, fewer animals at this dose level were available for teratological examination. Also, the duration of gestation was significantly increased (P , 0.05) for the high dose group. The NOEL for maternal toxicity was established at 5 mg/kg/day, based on decreased weight gain (mean) and an increased incidence of clinical signs at 25 and 75 mg/kg/day. Developmental effects included an increased number of fetuses and pups with micro- or anophthalmia and hepatic and diaphragmatic changes

There was an increased incidence of eye defects (microphthalmia or anophthalmia) in litters at 25 and 75 mg/kg/day, the increase being significant (P , 0.001) at 75 mg/kg/day (Table 2). The numbers of fetuses or pups with these eye defects also showed an increase: 3/583 (0.5%), 0/589 (0%), 5/589 (0.9%), and 13/110 (12%) at 0, 5, 25 and 75 mg/kg/day, respectively, significant at P , 0.001 at 75 mg/kg/day. The number of litters with microphthalmia at 25 mg/kg/day and at 75 mg/kg/day was significantly greater than the mean for the historical control (P , 0.05 and P , 0.001, respectively).

TABLE 1 Reproductive and Fetal Parameters Following Exposure of Fischer 344 Rats to Cyanazine by Gavage on Gestation Days 6 –15 a Dosage (mg/kg/day) Parameter No. females mated Females pregnant Pregnancy rate (%) No. females died C-section No. not pregnant No. pregnant No. of litters No. of litters with $1 resorption Mean litter size d Mean fetal wt. d (g) Male Female Combined Mean litter wt. d (g) Crown rump (cm) Natural delivery No. not pregnant No. pregnant No. of litters No. of litters with total resorption Mean litter size e Mean pup wt. e Mean litter wt. e (g) a

0

5

70 56 80 0

70 56 80 1b

70 62 89 0

7 25 25 7 (28%) 10.7 6 2.8

7 25 25 6 (24%) 11.6 6 1.6

6 25 25 11 (44%) 11.4 6 2.8

3.13 6 0.13 2.91 6 0.14 3.04 6 0.12 32.5 3.33 6 0.10 7 31 31 0 — 10.7 6 3.0 5.4 6 0.4 57.8

3.15 6 0.13 2.90 6 0.15 3.04 6 0.11 35.3 3.32 6 0.08 6 31 30 1 (3.2%) 10.4 6 2.8 5.5 6 0.4 57.2

Data are from Lochry (1985). Killed on day 25 (not pregnant). c Includes a nonpregnant rat which died on day 15 of presumed gestation. d Measured day 20 presumed gestation. e Measured day 1 postparturition. ** Significantly different from control at P , 0.01, Dunnett’s test. b

25

3.15 6 0.22 2.95 6 0.16 3.04 6 0.18 34.7 3.33 6 0.09 2 37 29 8 (22%) 10.9 6 2.3 5.5 6 0.4 60.0

75 70 53 76 13 c 8 21 21 20 (95%) 7.8 6 4.2 2.39 6 0.34** 2.23 6 0.31** 2.28 6 0.35** 17.8 3.04 6 0.21** 8 20 8 12 (60%) 7.0 6 2.4** 5.1 6 0.3 35.7

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TABLE 2 Occurrence of Microphthalmia or Anophthalmia in Litters (C-Section and Natural Delivery) of the Fischer 344 Rat Following Maternal Dosing with Cyanazine by Oral Gavage a Dosage (mg/kg/day) Eye malformation Microphthalmia Anophthalmia Combined

0b

5

25

75

2/55 (3.6%) 1/55 (1.8%) 2/55 c (3.6%)

0/55

2/51 (3.9%) 3/51 (5.9%) 4/51 c (7.8%)

4/16* (25%) 3/16* (19%) 7/16** (44%)

0/55 0/55

a

Data are from Lochry (1985). Historical control data showed 1/705 (0.14%) litters. c Includes cases of anophthalmia and microphthalmia in different pups within the same litter. * Significantly different from control at P , 0.05 (Fisher’s exact test). ** Significantly different from control at P , 0.001 (Fisher’s exact test).

period were variable for dams producing fetuses with eye defects (Table 4). Considering the 8- to 12-day period, the two untreated (control) dams producing litters with these eye malformations showed body weight gains which were similar to the group mean; at 25 mg/kg/day, two of four (50%) had higher body weight gains than the group mean and two were below; and at the high dose only two of the dams were affected more than the group mean, while five of seven demonstrated an increase in body weight gain compared to the group mean. Considering the entire dosing period (6 –15 days), a similar pattern was observed for all groups except 25 mg/kg/day, where two had body weight gains above the group mean, one below the group mean, and one equal to the group mean.

b

Characterization of Maternal Toxicity The eye malformations reported here in the Fisher 344 (F344) rat are thought to occur during days 8 –12 of gestation (Yoshitomi and Boorman, 1990). Maternal toxicity was assessed daily during this time period, as well as for the duration of the entire dosing period (6 –15 days). The parameters examined included absolute body weight, body weight gain, and clinical signs (Tables 3–5). The individual values of these parameters for those dams which gave rise to fetuses or pups with these eye malformations were compared with group mean values. Clinical signs were not reported daily, but only for the entire dosing period, 6 –25 days (Lochry, 1985). Maternal body weights. Following C-section, the body weights for dams in the 75 mg/kg/day group which gave birth to fetuses with anophthalmia or microphthalmia (Table 3) were consistently below the group means during days 8 –12. The dams allowed to deliver naturally showed variable body weights relative to the group means over this time period (days 8 –12). The two control dams had body weights which were just above and just below the group means, on each day; all (four) dams in the 25 mg/kg/day group had body weights which were above the mean on each day with the exception of dam No. 3 on day 9 which was below the mean; at 75 mg/kg/day, two dams (Nos. 7 and 10) weighed below the mean and two (Nos. 8 and 9) were above the mean on each day, except for day 11, when dam No. 8 was also slightly below the group mean body weight. Maternal body weight changes. The day-by-day maternal body weight changes during the 8- to 12-day

Clinical signs. Severe maternal toxicity, indicated by clinical signs of excitation such as hyperpnea, arched back, sensitivity to touch, and “tip-toe” walk, followed by evidence of depression, manifested as decreased motor activity, ataxia, ptosis, and death were observed in some animals (Table 5). Clinical signs of slight maternal toxicity included lacrimation, excess salivation, chromodacryorrhea, and urine-stained fur. The incidence of clinical signs in dams producing fetuses with eye defects was also examined at the level of the individual animal. An effort was made to determine whether those dams producing litters with malformed offspring displayed an abnormally high incidence of clinical signs of toxicity (Tables 5 and 6). No single severe clinical sign was associated with all of the affected dams. At the middose (25 mg/kg/day), three of the most common (slight) clinical signs were lacrimation (91%), excessive salivation (89%), and urine-stained fur (52%). For dams giving rise to offspring with eye malformations, the equivalent figures were as follows: lacrimation, 3 of 4 (75%); excessive salivation, 2 of 4 (50%); and urinestained fur, 1 of 4 (25%). Ptosis, a clinical sign of severe toxicity, was observed in 8 of 64 (13%) dams, while none of the 4 affected dams showed this sign. At 25 mg/kg/day, clinical signs of both slight and severe toxicity were observed less frequently in affected dams than the group mean. At 75 mg/kg/day, 13 rats died, including 1 which was nonpregnant. The majority of the surviving dams ($84%) displayed signs of slight toxicity. Ataxia with impaired or lost righting reflex, a sign of severe toxicity, was noted in 68% of animals at this dosage and in 2 of the 7 (29%) giving rise to offspring with eye malformations. In addition, 70% of the rats in this dose group displayed ptosis, including 6 of 7 (86%) dams producing offspring with eye malformations. Arched back was displayed by 41% of rats, with 4/7 (57%) for those giving rise to litters with eye malformations in offspring. At the high dose, ataxia was reported less frequently, but ptosis and arched

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TABLE 3 Maternal Body Weight (g) during Days 8 –12 of Gestation: Mean Values and Those for Individual Dams with Litters Showing Incidences of Anophthalmia or Microphthalmia a Mean maternal body weight (g 6 SD) Group (n)

Day 8

Day 9

Day 10

Day 11

Day 12

Control (56) 5 mg/kg/day (56) 25 mg/kg/day (62) 75 mg/kg/day

202 6 9.3 198 6 9.8* 189 6 9.0** 177 6 13** (n 5 51)

205 6 9.5 199 6 10** 190 6 8.7** 181 6 14** (n 5 44)

209 6 8.9 203 6 9.7** 191 6 9.3** 181 6 16** (n 5 44)

214 6 9.5 206 6 9.7** 191 6 10** 181 6 14** (n 5 43)

217 6 10 210 6 9.9** 194 6 9.9** 181 6 14** (n 5 43)

Day 10

Day 11

Day 12

216 208 195 202 200 212 165 190 207 156

216 211 192 202 199 216 168 179 197 178

220 214 199 202 212 213 164 182 193 177

161 162 179

175 167 172

176 167 175

Individual body weight Animal No.

Day 8

Day 9 Natural delivery

1 (Control) 2 (Control) 3 (25 mg/kg/day) 4 (25 mg/kg/day) 5 (25 mg/kg/day) 6 (25 mg/kg/day) 7 (75 mg/kg/day) 8 (75 mg/kg/day) 9 (75 mg/kg/day) 10 (75 mg/kg/day)

206 200 190 204 200 214 151 179 200 168

207 201 184 202 195 216 162 184 204 162 C-section

11 (75 mg/kg/day) 12 (75 mg/kg/day) 13 (75 mg/kg/day)

169 159 169

168 165 176

a Data are from Lochry (1985). * Significantly different from vehicle control at P , 0.05 (Dunnett’s test). ** Significantly different from vehicle control at P , 0.01 (Dunnett’s test).

back were reported more frequently in affected dams than the group mean. Overall, the clinical signs in dams giving rise to offspring with anophthalmia or microphthalmia were no worse than those in dams which did not produce offspring with these malformations. In another rat teratology study using F344 rats, the same eye defects were noted in a single fetus in the 20 control litters and in 5 fetuses in 3 of 20 litters at the highest dose, 25 mg/kg/day (Lu et al., 1981). Maternal toxicity was identified by comparing body weight gains of the dams from the treatment groups with the control group, as well as by observing the clinical signs exhibited. Examining these parameters of maternal toxicity during the crucial period of eye formation (days 8 to 12 of gestation) revealed a significant decrease in mean body weight gain: during days 6 to 12 of gestation, control body weight increased from 206 6 5.9 to 222 6 1.4 g and at 25 mg/kg/day from 204 6 5.1 to 209 6 1.4 g (P , 0.05, Dunnett’s test). However, the findings were not corroborated at the individual level. One affected dam

at 25 mg/kg/day gained as much body weight as the mean value of the control group (16 g) and substantially more than the group mean for this dose (5 g). Another dam gained 6.2 g, which was less than the control group mean but slightly more than the group mean for that dose. The third dam gained substantially less (23.3 g) than the group mean for the control or high-dose dams. The affected control dam had a body weight gain (14 g) which was slightly below the group mean. At the 25 mg/kg/day dose a single instance of clinical signs was recorded during the 6- to 12-day period: irritated swelling on the foot pads on day 8, which had cleared by day 10. This dam produced the most badly affected litter, containing fetuses with both microphthalmia (one fetus) and anophthalmia (two fetuses). Paradoxically, this individual had a body weight gain substantially higher than the group mean (and the same as the mean value for the control group). Thus, the contention that maternal toxicity (reduced body weight gain and increased clinical signs) played a major role in these fetal mal-

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TABLE 4 Maternal Body Weight Changes (g) during Days 8 –12 of Gestation: Mean Values and Those for Individual Dams with Litters Showing Incidences of Anophthalmia or Microphthalmia a Mean maternal body weight change (g 6 SD) Group (n)

Days 8–9

Days 9–10

Days 10–11

Days 11–12

Days 8–12

Days 6–15

Control (56) 5 mg/kg/day (56) 25 mg/kg/day (62) 75 mg/kg/day (44,43)

2.8 6 1.8 1.9 6 2.1* 0.8 6 4.1* 2.6 6 4.4

4.2 6 2.8 3.3 6 2.2 0.9 6 3.9** 0.2 6 6.0**

4.6 6 2.2 3.7 6 2.6 0.4 6 4.6** 0.0 6 8.0**

3.6 6 2.3 3.3 6 2.5* 2.6 6 4.5 20.7 6 4.9**

15 12 5 4

33 24** 8** 215**

Days 11–12

Days 8–12

Days 6–15

13 14 17 0 113 23 24 13 24 21

114 114 19 22 112 21 113 13 27 19

134 136 115 18 126 22 29 223 210 210

11 0 13

17 18 16

29 27 25

Individual body weight change Animal No.

Days 8–9

Days 9–10

Days 10–11 Natural delivery

1 (Control) 2 (Control) 3 (25 mg/kg/day) 4 (25 mg/kg/day) 5 (25 mg/kg/day) 6 (25 mg/kg/day) 7 (75 mg/kg/day) 8 (75 mg/kg/day) 9 (75 mg/kg/day) 10 (75 mg/kg/day)

11 11 26 22 25 12 111 15 14 26

17 19 111 0 15 24 13 16 13 26

13 0 23 0 21 14 13 211 210 122 C-section

11 (75 mg/kg/day) 12 (75 mg/kg/day) 13 (75 mg/kg/day)

21 16 17

27 23 13

114 15 27

a

Data are from Lochry (1985). * Significantly different from control at P , 0.05 (Dunnett’s test). ** Significantly different from vehicle control at P , 0.01 (Dunnett’s test).

formations received only equivocal support at the level of the individual dam. Rabbit Teratology

Cyanazine was given by oral gavage to 22 mated NZW female rabbits/group at 0, 1.0, 2.0, or 4.0 mg/ kg/day on days 6 –18 of gestation (Dix et al., 1982). Slight maternal toxicity was reported at 4 mg/kg/day and possibly 2 mg/kg/day (decreased food consumption and weight gain). Fetal effects at 4 mg/kg/day included decreased litter size, increased resorptions, increased incidence of a 13th rib, and a complete loss of litter in 5/20 dams. At 2 mg/kg/day, slight increases in mean resorptions/dam (0.8 in control vs 1.2, treated) and 13th rib were observed, in addition to 1/22 maternal deaths and 1/22 abortions. The NOEL value for maternal and developmental toxicity was 1 mg/kg/day. Malformations such as eye defects (microphthalmia in 1 fetus) and thoracoschisis along with microphthalmia (1 fetus) were observed in two different litters at 4 mg/kg/day. These effects were accompanied by maternal toxicity, indicated by

a loss of body weight of 6.4 and 6.8%, respectively, over the entire dosing period, compared with a mean body weight gain in controls of 1.2% and a group mean loss of 2.5% at 4 mg/kg/day. Eye formation in the rabbit embryo occurs during the 8- to 12-day period of gestation (Edwards, 1968). It is possible that this is the key time period during which agents acting on eye development exert their effects. In the present study, during the 6- to 12-day period of gestation (time period closest to critical period available in the report), the dam producing the fetus with microphthalmia had a body weight gain of 1.2%, compared with the control group mean body weight gain of 1.1% and the high-dose group mean loss of 1.0%. The dam which gave rise to the fetus with thoracoschisis and microphthalmia had a body weight loss of 0.14%, which is also less than the group mean (21.0%) for the high dose. It appears that in this case the weight loss in the affected dams occurred between day 12 and day 18, i.e., after the critical period of eye development for this species. Hence, the timing of the maternal toxicity described does not correlate with the timing of eye development.

MATERNAL INFLUENCE ON TERATOGENICITY: CYANAZINE

TABLE 5 Individual Litter Data Showing Types of Eye Defects and Severity of Maternal Clinical Signs a Animal No.

Affected fetuses

Microphthalmia

Anophthalmia

Clinical signs b

2 1 2 1 1 1 1 1 2 1

No No Slight c Slight Slight Slight Severe d Severe Severe Severe

2 2 2

Severe Severe Slight

Natural delivery 1 (Control) 2 (Control) 3 (25 mg/kg) 4 (25 mg/kg/day) 5 (25 mg/kg/day) 6 (25 mg/kg/day) 7 (75 mg/kg/day) 8 (75 mg/kg/day) 9 (75 mg/kg/day) 10 (75 mg/kg/day)

1/13 2/12 1/11 1/10 2/9 1/12 2/3 1/4 1/8 3/5

1 1 1 2 1 2 2 2 1 2 C-section

11 (75 mg/kg/day) 12 (75 mg/kg/day) 13 (75 mg/kg/day)

3/12 2/10 1/5

1 1 1

a

Data are from Lochry (1985). Clinical signs observed from day 6 to day 25 of presumed gestation; data specifically for days 8 to 12 are not available in Lochry (1985). c For slight clinical signs, dams showed one or more of lacrimation, salivation, and/or urine-stained fur. d For severe clinical signs, dams showed one or more of ataxia, ptosis, and/or arched back. b

DISCUSSION

In a developmental toxicity study of cyanazine in the F344 rat (Lochry, 1985), fetal eye malformations were noted at the high (75 mg/kg/day) and mid (25 mg/kg/ day) dosages. The low dosage (5 mg/kg/day) was therefore determined to be the developmental NOEL. Decreases in mean maternal body weight gains and increased clinical signs of toxicity at both the high and mid dosages resulted in a maternal NOEL of 5 mg/kg/ day. Generally, in order to determine whether or not the conceptus is uniquely susceptible, the developmental and maternal NOEL values are compared. The A/D ratio (adult NOEL:developmental NOEL) has been advocated previously as an index of comparative teratogenic hazard (Johnson, 1981) and has been used to characterize the developmental effects of chemicals. In the current study an A/D ratio of 1 for both the rat and the rabbit and the observation of fetal malformations along with maternal effects are suggestive of maternal toxicity. Reviewing the literature it appears that the strategy of carefully characterizing the observed maternal toxicity at the individual level is often not employed. To determine if the malformations observed were the result of maternal toxicity, two approaches were adopted: individual vs group mean data were considered and data were examined during the specific period of gestation when the developmental malformations were likely to have occurred.

93

The maternal toxicity of cyanazine was characterized by examining specific parameters for each dosage group, e.g., mean values of body weight, body weight gain, and clinical signs. The reported maternal toxicity is unlikely to be the principal cause of the developmental/fetal defect since the group mean data were not confirmed at the individual level, i.e., in those dams producing offspring with eye malformations. Dams bearing litters with fetal malformations showed signs of toxicity no worse than the dams producing litters with no fetal malformations. A similar pattern of effects was noted with cyanazine in an earlier study (Lu et al., 1981). The repeated occurrence of the same set of malformations in independent studies (Lu et al., 1981; Lochry, 1985) strengthens the case for direct developmental toxic potential rather than the malformations being a consequence of maternal toxicity. Furthermore, the finding extends to a second species: In the developmental toxicity study of cyanazine in the New Zealand rabbit (Dix et al., 1982), the cases of microphthalmia were not accompanied by toxicity in the individual dams during the period of eye development. The group data, however, suggested possible maternal toxicity in the form of reduced mean body weight gain during this time period. Information on maternal toxicity can be obtained for various time points and evaluated during the following periods: (i) overall gestation, (ii) organogenesis, (iii) specific periods of organogenesis, and/or (iv) exposure. To demonstrate the maternal–fetal involvement, relating the day or stage of pregnancy or fetal development to the time of occurrence of maternotoxic signs has been recommended (Khera, 1987). The rationale for selecting one period of gestation over another may be based on knowledge of the defect and its associated embryology. It has been stated, “Overall the evidence accumulated . . . suggests that dosimetry–teratogenicity determinants are quite distinct for the developmental phase during which a particular organ differentiates and a chemical teratogen acts upon the embryo” (Welsch, 1995). For example, specific periods during gestation in the rat and the mouse were studied to assess the role of maternal toxicity in the production of supernumerary ribs following chemical exposure (Chernoff et al., 1987; Kavlock et al., 1985). An analogous approach was used here: Maternal toxicity was assessed during the period of fetal eye development in the rat and rabbit. In this way, the association between maternal toxicity and fetal malformations could be determined more precisely. For the F344 rat and the NZW rabbit, the toxic effects in the dams giving rise to malformed offspring were no worse for this period than those for dams producing unaffected offspring. The etiology of eye defects could be multifactorial in nature, possessing both genetic and environmental components. Mutations in the gene for the transcription factor, Pax6, induce marked developmental abnormalities in the central nervous system and the eye as

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TABLE 6 Clinical Signs Observed in Pregnant Rats: Group Means and Individual Data for Dams Producing Offspring with Eye Malformations a Dosage 25 mg/kg/day Clinical sign b Slight Lacrimation Salivation Urine-stained fur Severe Ataxia e Ptosis f Arched back

75 mg/kg/day

Group mean c

Affected dams d

Group mean c

Affected dams d

58/64 (91%) 57/64 (89%) 33/64 (52%)

3/4 (75%) 2/4 (50%) 1/4 (25%)

54/56 (96%) 47/56 (84%) 54/56 (96%)

7/7 (100%) 6/7 (86%) 6/7 (86%)

0/4 0/4 0/4

38/56 (68%) 39/56 (70%) 23/56 (41%)

2/7 (29%) 6/7 (86%) 4/7 (57%)

0/64 8/64 (13%) 0/64

a

Data are from Lochry (1985). Observed during days 6 –25 of gestation; clinical signs data specifically for days 8 –12 not available. c Includes dams producing litters with eye malformations in fetuses or pups. d Dams producing litters with eye malformations in fetuses or pups. e Includes lost or impaired righting reflex. f Includes instances of decreased palpebral size. b

noted in Smalleye, the Pax6 mutant mouse (Stoykova et al., 1997). Pax6 is the transcription activator that regulates eye development across species spanning from Drosophila to man (Tang et al., 1998). In developmental toxicity studies submitted for regulatory purposes under FIFRA, the genetic component is rarely explored. In the absence of a known genetic component, other factors that can be considered in the evaluation of a chemical’s developmental toxicity include historical control data, information from reproductive toxicity studies, and information from chemical analogs (structure–activity relationships). Congenital anophthalmia rarely occurs in the F344 rat (Yoshitomi and Boorman, 1990). Historical control data for the F344 rat used by Lochry (1985) indicated a very low incidence: 1/705 (0.14%) litters and 1/9183 (0.01%) fetuses, both of which are well below the incidences reported in the study (Table 2). In a two-generation rat reproduction study for cyanazine (Nemec, 1987), the NOEL for pups (reduced pup body weight and viability) was 75 ppm (5.6 mg/kg/day) and for reduced parental body weight was 150 ppm (11.2 mg/kg/day), suggesting that the pup was more sensitive to the toxic effects of cyanazine than the dam. No eye defects were observed. However, the absorbed dosages in the reproduction study (diet) were much lower than those in the developmental toxicity (gavage) study. Also, close structural analogs of cyanazine, the triazine pesticides cyromazine, atrazine, and simazine, caused eye malformations in offspring of the F344 rat or NZW rabbit following maternal dosing (Arthur, 1984; Arthur and Infurna, 1984; Mainiero et al., 1986; Nemec, 1985, 1986). In a teratology study on Sprague–Dawley rats (Lu, 1983) such malformations were not noted, suggesting that the F344 rat may be more sensitive to triazine pesticides.

Since the study with the most sensitive species/strain usually drives the risk assessment, in the absence of data documenting that the F344 rat is not a suitable animal model for extrapolation to humans, these findings will be considered critical data. Developmental effects occurring at maternally toxic dose levels have been inconsistently interpreted, e.g., dose-related incidence of supernumerary ribs in the absence of other findings was considered to be (i) of no teratogenic significance (Khera, 1974; Hudak and Ungary, 1978) or (ii) suggestive of teratogenic activity (Basford and Fink, 1968; Yasuda and Maeda, 1973). Current information is inadequate to assume that developmental toxicity at maternally toxic doses results predominantly from maternal toxicity; it may be that the dam and developing organism are sensitive at the same dose level. Previous reviews on the subject have suggested that if an agent does act directly on the embryo, albeit at the same dosage that affects the pregnant animal, then it remains a potential human hazard (Daston, 1994). Additionally, mere correlation of maternal toxicity with fetal effects does not imply causality (Chernoff et al., 1987). While maternal influence is possible, it may not necessarily be the underlying mechanism of action. Furthermore, the severity of the effect on the fetus needs to be considered; i.e., the effect may be severe/life-threatening, while the maternal effects such as slight weight loss are minor or transient. Another confounding factor is that maternal effects may be reversible, whereas effects on the developing fetus may be permanent, underscoring the importance of characterization of the maternal effects. Examining the data in this manner leads to a more exacting interpretation of teratogenic potential.

MATERNAL INFLUENCE ON TERATOGENICITY: CYANAZINE

CONCLUSIONS

The weight-of-evidence approach was used in determining the developmental toxic potential for cyanazine. ● Eye malformations occurred in a dose-related manner at both mid dose and high dose with differing levels of maternal toxicity. ● Similar outcome was confirmed in two separate studies. ● Supportive evidence was noted in another species (rabbit). ● Eye malformations have been noted in developmental toxicity studies with other triazine pesticides.

Examining group mean data during the entire period of gestation, the developmental toxicity of cyanazine appeared to be associated with, or possibly be a secondary consequence of, the reported maternal toxicity. However, under closer scrutiny, by focussing on individual data during the discrete time period(s) in which particular fetal malformations were most likely to have occurred, it appeared that developmental toxicity was independent of maternal toxicity. The teratogenic effects of cyanazine may be exacerbated by maternal toxicity, but are not a direct consequence of maternal toxicity. A more critical evaluation that verifies the apparent maternal toxicity at the individual level thus may help in determining the developmental toxic potential of a chemical. REFERENCES Arthur, A. T. (1984). A Teratology Study of Atrazine Technical in New Zealand White Rabbits. Ciba-Geigy, Study No. 68 – 84, DPR Vol. 220 – 050, 2118 No. 016885, 072135. Arthur, A. T., and Infurna, R. N. (1984). A Teratology Study of Simazine Technical in New Zealand White Rabbits. Ciba-Geigy, Study No. 62-83, DPR. Vol. 213– 044, Report No. 20194. Basford, A. B., and Fink, B. R. (1968). The teratogenicity of halothane in the rat. Anaesthesiology 29, 1167–1173. Chernoff, N., Kavlock, R. J., Beyer, P. E., and Miller, D. (1987). The potential relationship of maternal toxicity, general stress, and fetal outcome. Teratogen. Carcinog. Mutagen. 7, 241–253. Daston, G. P. (1994). Relationships between maternal and developmental toxicity. In Developmental Toxicology (C. A. Kimmel and J. Buelke-Sam, Eds.), 2nd ed., pp. 189 –212. Raven Press, New York. Dix, K. M., Cassidy, S. L., and Daniel, D. L. (1982). Teratology Study in New Zealand White Rabbits Given Bladex Orally, Shell Oil Company SBGR.82.357, DPR Vol. 307– 045 No. 38545. Edwards, J. A. (1968). The external development of the rabbit and rat embryo. In Advances in Teratology (D. H. M. Woollam, Ed.), pp. 246 –250. Academic Press, New York. Hudak, A., and Ungvary, G. (1978). Embryotoxic effects of benzene and its methyl derivatives. Toxicology 11, 55– 63. Iyer, P., Gammon, D., Gee, J., and Pfeifer, K. (1995). Characterization of maternal toxicity in the teratogenicity of the herbicide cyanazine. Int. Toxicol. 74-P-8. Johnson, E. M. (1981). Screening for teratogenic hazards: Nature of the problems. Annu. Rev. Pharmacol. Toxicol. 21, 417– 429.

95

Kavlock, R. J., Chernoff, N., and Rogers, E. H. (1985). The effect of acute maternal toxicity on fetal development in the mouse. Teratogen. Carcinog. Mutagen. 5, 3–13. Khera, K. S. (1974). Teratogenicity and dominant lethal studies on hexachlorobenzene in rats. Food Cosmet. Toxicol. 12, 471– 473. Khera, K. S. (1981). Fetal aberrations and their teratological significance. Fundam. Appl. Toxicol. 1, 13–18. Khera, K. S. (1987). Maternal toxicity in humans and animals: Effects on fetal development and criteria for detection. Teratogen. Carcinog. Mutagen. 7, 287–295. Kimmel, C. A., and Wilson, J. G. (1973). Skeletal deviations in rats—Malformations or variations? Teratology 8, 309 –315. Lochry, E. A. (1985). Study of the Developmental Toxicity of Technical Bladex Herbicide (SD 15418) in Fischer 344 Rats, Argus Research Laboratories, Inc. Protocol 619-002, Shell Oil Company, DPR Vol. 307– 027 No. 27089. Lu, C. C., Tang, B. S., and Chai, E. Y. (1981). Technical Bladex (SD 15418) Teratology in Rats, Shell Westhollow Research Center, WRC RIR-180, DPR 307-045 No. 38544. Lu, C. C. (1983). Teratologic Evaluation of Bladex in SD CD Rats, Research Triangle Institute, RTI Project No. 31T-2564, Shell Oil Company, WRC RIR-311, DPR Vol. 307-045 No. 38546. Mainiero, J., Eimbert, K., Wright, J., Infurna, R. N., Arthur, A. T., and Yau, E. T. (1986). Simazine Technical: A Teratology Study in Rats. Ciba-Geigy Study No. 83658, DPR Vol. 213-065, Report No. 67847. Nemec, M. D. (1985). Larvadex Technical: Teratology Study in Rabbits, Ciba-Geigy Corp., Report Wil-72662, DPR Vol. 414 – 034, Report No. 031090. Nemec, M. D. (1986). A Teratology and Post-natal Study in Albino Rabbits with Cyromazine Technical, Report No. Wil-82008, CibaGeigy Corp., DPR Vol. 414 – 064, Report No. 055966. Nemec, M. D. (1987). Two-Generation Reproduction Study of Technical Bladex Herbicide (SD 15418) in Rats, WIL Research Laboratories, Inc., WIL-93001, DuPont SRO 15– 87, DPR Vol. 307-062 No. 61221. Palmer, A. K., and Kavlock, R. J. (1987). Consensus workshop on the evaluation of maternal and developmental toxicity work group II report: Study design considerations. Teratogen. Carcinog. Mutagen. 7, 311–319. Schardein, J. (1987). Approaches to defining the relationship of maternal and developmental toxicity. Teratogen. Carcinog. Mutagen. 7, 255–271. Stoykova, A., Gotz, M., Gruss, P., and Price, J. (1997). Pax6-dependent regulation of adhesive patterning, R-cadherin expression and boundary formation in developing forebrain. Development 124(19), 3765–3777. Tang, H. K., Singh, S., and Saunders, G. F. (1998). Dissection of the transactivation function of the transcription factor encoded by the eye developmental gene PAX6. J. Biol. Chem. 273(13), 7210 –7221. USEPA (1982). Pesticide Assessment Guidelines, Sub-division F, Hazard Evaluation: Human and Domestic Animals EPA-540/9-820025. Welsch, F. (1995). Pharmacokinetics in developmental toxicology: 2-Methoxyethanol as a prototype chemical for teratogenicity studies at different stages of gestation. CIIT Act. 15(1), 1. Yasuda, M., and Maeda, H. (1973). The significance of the lumbar rib as an indicator in teratogenicity tests. Senten Ijo 13, 25–29. Yoshitomi, K., and Boorman, G. A. (1990). In Pathology of the Fischer Rat (G. A. Boorman, S. L. Eustis, M. R. Elwell, C. A. Montgomery, Jr., and W. F. MacKenzie, Eds.), p. 244. Academic Press, San Diego.