1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18 Jul 1984 to 04 Oct 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-chloroethoxy)-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)carbamoyl]benzene-1-sulfonamide

EC Number:

617-298-9

Cas Number:

82097-50-5

Molecular formula:

C14H16ClN5O5S

IUPAC Name:

2-(2-chloroethoxy)-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)carbamoyl]benzene-1-sulfonamide

Test animals

Species:

rat

Strain:

other: Tif: RAif(SPF), hybrids of RII/1 x RII/2

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: about 2 months, nulliparous
- Weight at study initiation: 180 - 200 g
- Housing: The females were placed in groups of four in Macrolon cages
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: Acclimation under test conditions did take place during the period of time between allocation to the corresponding group on day 0 and the first treatment on day 6 post coitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-22°C
- Humidity: 45-65%
- Air changes: 16-20 air changes/hour
- Photoperiod: 12 hours light per day (6 a.m. -6 p.m. )

IN-LIFE DATES: From: 18 Jul 1984 To: 04 Oct 1984

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous solution of a sodium-carboxymethyl cellulose

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: daily
- Mixing appropriate amounts with vehicle: The test item was suspended in an aqueous solution of 0.5 % carboxymethyl cellulose (CMC) and administered orally by gavage from day 6 until day 15 of pregnancy, inclusive. The suspension of the test material was freshly prepared daily and the homogeneity of the suspension was maintained by means of a magnetic stirrer. The volume administered was adjusted to 10 mL/kg of actual daily body weight. The stability of the test article in the above suspension for at least two hours was ascertained by the sponsor. The solutions prepared contained 10 mg/mL, 30 mg/mL and 90 mg/mL compound of the test item

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: each cage was subdivided in two parts by a damper, separating the sexes until about 3 a.m. when the damper opened by means of an automatic device. At 7 a.m. to 9 a.m., successful mating was determined
- M/F ratio per cage: 3 females to 1 male
- Proof of pregnancy: either by the presence of a vaginal plug or by the presence of spermatozoa in the vaginal smear. This day was designated day "0" of pregnancy.

Duration of treatment / exposure:

From day 6 until day 15 of pregnancy, inclusive

Frequency of treatment:

Daily

Duration of test:

Until day 21 p.c.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low dose group; Group 2

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

Intermediate dose; Group 3

Doses / concentrations 3

Dose / conc.:

900 mg/kg bw/day

Remarks:

High dose; Group 4

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose-levels were selected on the basis of a previously conducted preliminary study. Based on these data, the dose-levels, previously proposed to be 200, 600 and 1200 mg/kg of actual daily body weight, were reduced to 100, 300 and 900 mg/kg for the main study by amendment in order to obtain a no-effect-level, an intermediate dose-level and a toxic effect-level

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily for mortality, signs of abortion and general condition

BODY WEIGHT:
- Time schedule for examinations: daily

FOOD CONSUMPTION:
- Time schedule: days 6, 11, 16 and 21 of pregnancy.
The mean daily food consumption was calculated as follows: (food consumption (g) per period)/ (days per period x no. of animals per cage)

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day: Dams were killed by carbon dioxide inhalation (dry ice) on day 21 of pregnancy and fetuses were removed by Cesarean section. Dams which died spontaneously during the experimental phase were subjected to macroscopic pathological examination. The number of implantations were also recorded.
-Record of macroscopic pathological examination of the main organs of the thoracic and abdominal cavities, in particular the genitals.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one thirds per litter
- Skeletal examinations: Yes: two thirds per litter
- Head examinations: Yes: all per litter
- Anogenital distance of all live rodent pups: yes

The live fetuses were subjected to careful external inspection, special attention being attached to the following body regions: extremities or parts thereof, tail, head, brain, eyes, pinnae, oral orifice, cleft lip and/or cleft jaws and/or cleft palate, all body regions (e.g. generalized or localized oedema, haemorrhage).
The viscera of one third of the fetuses per litter were examined according to the slicing technique of Wilson: A solution containing ethyl alcohol, formaldehyde and acetic acid served as a fixative. Before slicing the fetuses were treated with an ammonium hydrogen carbonate solution and rinsed in tap water. All specimens were finally stored individually, i.e. per fetus, in a mixture of ethanol and glycerol. In case of eventual discrepancy in sex-determination between external inspection and slicing technique, the sex was corrected in accordance with the results of the latter.
Skeletal assessment in two thirds of the fetuses per litter was accomplished following staining according to the technique of Dawson: After clearing with potassium hydroxide and staining with alizarin reds, the specimens were, by several steps, finally passed into glycerol. This type of examination covered, apart from revealing possible instances of skeletal anomalies and/or malformations, also the state of skeletal maturation of the fetuses shortly before term on day 21 post coitum.

Statistics:

General:
All values listed in the statistics section of this report were analysed by the Student's t-test, except for the analysis of fetal anomalies, which were analysed by the CHI-square test. Statistical analysis is performed to draw attention to distinct values. A statistically significant difference between two values does not necessarily imply biological relevance of that deviation and is not conclusive for a treatment related effect. Hence, the responsible scientist may not comment on statistically significant values lying within the physiological range and on the other hand may comment on statistically not significant values, which differ substantially from the expected normal values.

Explanation of signs and remarks
* significant at p=0.05 (t-test)
** significant at p=0.01 (t-test)
+ significant at p=0.05 (CHI-square test)
++ significant at p=0.01 (CHI-square test)
+++ significant at p=0.001 (CHI-square test)
N number of samples or number of animals

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in thecontrol group died spontaneously on day 19 post coitum, i.e. four days after the end of the treatment period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain, determined on the basis of the individual actual daily body weight, was not altered in the low-dose group (100 mg/kg bw/day) in comparison with the vehicle control. The intermediate and high-dose groups (300 and 900 mg/kg bw/day, respectively) reacted to the treatment by a significant and dose-related reduction of body weight gain (based on the daily group means) during the time of administration of the test article (see Table 1). The most significant effect was observed for the first phase of administration i.e. between days 6 and 11 p.c.The females of the high-dose group were still affected in this respect during the post-treatment period.
With regard to the average "corrected body weight gain at necropsy", no statistical difference was found for either dose group in comparison with the vehicle control. At the high-dose level (900 mg/kg bw/day) some reduction was indicated, however.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption during the treatment period was found to be reduced in a dose-related manner for the intermediate and high-dose groups (300 and 900 mg/kg bw/day) in comparison with the vehicle control. The reduction in food intake of both groups was marked and statistically significant in the first period of administration of the test item between days 6 and 11 post coitum. In contrast, food consumption was significantly raised in the high-dose group between days 16 and 21 p.c., i.e. during the post-dosing period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Neither external inspection nor macroscopic pathological examination of the dams' organs, in particular of the thoracic and abdominal cavities, did reveal any obvious alterations at sacrifice shortly before term on day 21 post coiturn.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No partial abortion or was observed in any of the four groups, and none of the pregnant females showed total abortion, early birth of the whole litter or total resorption of the litter.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The pregnancy rates expressed as average number of implantation sites per female were comparable for all groups in the present experiment. Between 87.5% and 100% of the mated females became pregnant (implantations other than deciduomata). The slightly increased post-implantation loss observed in the high-dose group is based on the resorption rate. The mean litter size was comparable for all dose groups and the vehicle control.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Partial resorption of the litter occurred in all four groups. The number of females with at least one resorption site was between 9 (intermediate dose group) and 11 (low and high-dose group).

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

The number of early resorptions (total as well as relative, i.e. in per cent of the number of implantation sites) was comparable for the low and intermediate dose groups in comparison with the vehicle control; a marginally increased rate was noted for the high-dose group (Chiz -test, Yates' correction: observed p > 0.03). Late resorption were rare. One instance each was recorded for the low and high-dose group, respectively.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses were found in any of the four experimental groups.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

Early parturition of part of the litter (12 out of 17 fetuses) was noted for one dam of the vehicle control group, no experimental significance being attached to this finding, however.

Changes in number of pregnant:

not specified

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

Maternal systemic toxicity

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

2

Dose descriptor:

NOAEL

Remarks:

Maternal developmental toxicity

Effect level:

900 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average body weight of the live male, of the live female and of all the live fetuses was not altered for the low and intermediate dose group, respectively, in comparison with the vehicle control. In the high-dose group, however, fetal body weights were diminished significantly in either sex.

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The average number of male and female fetuses per litter was similar for the four groups. The male-to-female sex ratios were comparable for all groups (Chiz -test, Yates' correction, observed p > 0.1).

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

not specified

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

The gross inspection of the live fetuses revealed the following instance of malformation in the intermediate dose group (cf. also "Fetal skeletal examination"): Group 3, no. 65/R 02 (sex undetermined), generalized edema, maxillary bypoplasia, dysgenesis of posterior part of body
(rudimentary bind-limbs). No other external anomalies and/or malformations were recorded in either the dose groups or in the vehicle control.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The evaluation of the fetal skeletons (see Table 4) revealed one fetus from the intermediate dose group displaying multiple skeletal malformations (cf. also "Fetal external findings"): group 3: no. 65/R 02 (sex undeterained), Nasals absent, frontals partially absent, ischia, feaora, tibiae and fibulae shortened. Additional anomalies: Sternebrae 2,4 bipartite, “wide suture”. Further skeletal anomalies were found in the dose groups as well as in the vehicle control group. The incidence of sternebral anomalies in the high-dose group was slightly but significantly raised in comparison with the vehicle control (7.0% and 2.3%, respectively).

Concerning skeletal maturation of the fetuses at sacrifice of the mother animals near term on day 21 post coitum, a delay of ossification (in particular: phalangeal nuclei of hind-limbs, cervical and thoracic vertebral centres) was recorded for the high-dose group. The intermediate dose group was concerned only marginally in this respect (e.g. cervical vertebral centres); this finding is not considered to be of an experimental significance.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Carrying out the slicing technique for "visceral examination", one anomaly was recorded for the vehicle control: group 1: no. 14/R 05 (fem.), Dilatation of renal pelvis (bilateral) Concerning malformations, two instances were found in the highdose group: group 4: no. 84/L 04 Cfea.), no. 96/R 07 (male), Anopbthalmia (right side) Encephalocele. No additional instances of of anomalies and/or malformations were found in any fetus examined from either experimental group

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: Sternebral anomalies

Description (incidence and severity):

Slightly but significantly raised In the high-dose group, in comparison with the vehicle control (7.0% and 2.3%, respectively).

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

900 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1: Mean body weights of dams (in g)

	Group 1	Group 2	Group 3	Group 4
Dose (mg/kg)	0	100	300	900
Day:
0	193.6	195.1	193.8	192.4
1	199.8	202.2	201.1	201.9
2	207. 7	210 .2	208.4	207.9
3	212.8	215.0	212.7	214.2
4	217.4	222.3	217. 8	218. 8
5	222.8	226. 0	222.7	224.2
6	227.7	23 0 .5	226.7	227.5
7	231.5	232.1	225.8	221.7*
8	236.7	23 8 .6	23 0. 6*	227.5*
9	243 .1	243. 6	237.5	229.0*
10	249.7	250.4	241.4*	23 2. 9*
11	256.6	256.7	248.8*	237.6*
12	260 .9	261.8	253.4*	243.9*
13	268.9	268.1	259. 8*	249.2*
14	275.3	274.9	265.5*	256.3*
15	284.6	284. 5	274. 8*	261.9*
16	296.0	295.6	284.4*	273 .9*
17	310.5	311.4	301.6	290. 2*
18	326.8	326.9	316.9	307.1 *
19	342.6	343. 5	331.7	322.6*
20	356.9	356.6	345.3	336.9*
21	361.9	363.4	353. 8	346. 0

 

Table 2: Summary of cesarean section, pregnancy data

dose (mg/kg)	Group 1	Group 2	Group 3	Group 4
	0	100	300	900
mated dams	24	24	24	24
dams sacrificed on day 21 p.c.	23	24	24	24
dams w deciduomata	0	0	0	2
pregnant females	23	24	22	21
%	95.8	100	91.7	87.5
pregnant females abortion	0	0	0	0
pregnant females w partial prernat. birth at day 21	1	0	0	0
%	4.2	0	0	0
pregnant females w total resorp.	0	0	0	0
%	0	0	0	0
pregnant females w part. resorp.	10	11	9	11
%	41.7	45.8	37.5	45.8
pregnant females w live fetuses	22	24	22	21
%	91.7	100	91.7	87.5

 

Table 3: Summary of cesarean section, uterine data

dose (mg/kg)	group 1	group 2	group 3	group 4
	0	100	300	900
mated dams	24	24	24	24
pregnant females alive at day 21	22	24	22	21
pregnant females w live fetuses	22	24	22	21
Implant.sites total no	340	372	324	326
mean no	15.5	15.5	14.7	15.5
Early resorp total no	13	18	13	26
mean no	0.59	0.75	0.59	1.24
%	3.8	4.8	4.0	8.0
Late resorp. total no	0	1	0	1
mean no		0.04		0.04
%		0.3		0.3
Total resorp. Total no	13	19	13	27
Mean no	0.59	0.79	0.59	1.29
%	3.8	5.1	4.0	8.3
Dead fetuses Total no	0	0	0	0
Mean no
%
All dead implants Total no	0	0	0	0
Mean no
%
Post-implantation loss (%)	3.8	5.1	4.0	8.3

Table 4: Summary of fetal skeletal anomalies and/or malformations

dose (mg/kg)	Group 1	Group 2	Group 3	Group 4
	0	100	300	900
litters evaluated	22	24	22	21
fetuses examined	218	234	210	199
skeletally malf. fetuses : total	0	0	1	0
(%)	0	0	0.48	0
per litter	0	0	0.05	0
( %)	0	0	0.45	0
litters w skeletally malformed fetuses: total	0	0	1	0
( % )	0	0	4.5	0
Fetuses w skeletal anomalies: total	5	3	4	14++
( % )	2.29	1.28	1. 90	7.04
per litter	0.23	0.13	0.18	0.67
( % )	1.32	1.22	1.28	7.91
Litters w skel. Anomalous fetuses: Total	3	2	4	7
( % )	13.6	8.33	18.2	33.3
fetuses w delayed skel. Maturation: total	218	233	210	199
( % )	100	99.5	100	100
Per litter	9.91	9.71	9.55	9.48
( % )	100	99.5	100	100
litters w delayed . skel. Maturation: total	22	24	22	21
(%)	100	100	100	100

 

Applicant's summary and conclusion

Conclusions:

To summarize, test item was devoid of an embryotoxic activity and teratogenic potency in the rat under the present experimental conditions at the doses of 100 and 300 mg/kg day. At 900 mg/kg, a dose close to the limit test (1000 mg/kg), abnormal ossification of fetal sternebrae was slightly raised. This marginal effect was associated with maternal toxicity and fetotoxicity, i.e. marked delay of physiological growth of the fetuses.

Executive summary:

In this OECD 414 study, performed under GLP, the test item was evaluated for its possible teratogenic potency in the rat at dose-levels causing maternal and/or embryonic toxicity. Based on the results of a previously conducted preliminary teratology study, the daily doses were selected at 0, 100, 300 and 900 mg/kg of body weight. The test material was suspended in a 0.5% aqueous solution of sodium-carboxymethylcellulose (CMC) and administered once daily orally by intubation from day 6 until day 15 of pregnancy, inclusive. Body weights and clinical signs of the dams were recorded daily, food consumption was determined at least once weekly. Dams were killed a short time prior to expected delivery and submitted to macroscopic pathological examination. The uteri were dissected and contents examined. Live fetuses were weighed, sexed and evaluated for external, visceral and skeletal abnormalities. During the period of administration the pregnant females of the intermediate and high-dose groups reacted to the treatment by a dose-related depression of the body weight development. Food intake was also significantly reduced in the high-dose group during the first part of the treatment period; the intermediate dose group was affected to a lesser extent in this respect. At sacrifice, no pathological alterations were revealed in the dams of either group, except for two females of the high-dose group showing deciduomata only. Pregnancy rates as well as average litter size were comparable for the dose groups and the vehicle control. A marginal increase of the resorption rate was recorded for the high-dose group. The live fetuses of the low and intermediate dose groups remained unaffected by the treatment. The male-to-female sex ratio was not altered in any dose group. The average body weight of the live fetuses was not impaired in the low and intermediate dose group. No obvious alterations of the skeletal maturation of the live fetuses was recorded for the low and intermediate dose groups. The progeny of the high-dose group displayed an evident delay of physiological growth as indicated-by a significantly diminished body weight and a high number of still incompletely ossified skeletal elements. The skeletal anomalies, in particular the instances of sternebral anomalies recorded for the vehicle control, the low-dose and the intermediate dose groups, were not considered to be of experimental significance. However, the incidence of sternebral anomalies in the high-dose group was slightly but significantly raised in comparison with the vehicle control (7.0% and 2.3%, respectively).

In conclusion, the test item was devoid of an embryotoxic activity and teratogenic potency in the rat under the present experimental conditions at the doses of 100 and 300 mg/kg day. At 900 mg/kg, a dose close to the limit test (1000 mg/kg), abnormal ossification of fetal sternebrae was slightly raised. This marginal effect was associated with maternal toxicity and fetotoxicity, i.e. marked delay of physiological growth of the fetuses.