pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity findings regarding reprotoxic toxicity:

-      3 months feeding study in Beagle dogs: reduced testis weights in dogs exposed to 2500 ppm for 3 months, dilatation of prostatic tubuli (reported as cystic dilatation of glandular tissue) and minimal tubular atrophy of the testis or reduced spermatogenesis at 500 ppm and 2500 ppm (Altmann 1992).

-      1 year feeding study in Beagle dogs: reduction of the testis weight was statistically significant at 200 and 1000 ppm. Furthermore, unilateral tubular atrophy in one male; bilateral occurrence of spermatic giant cells in the testicular spermatogenic epithelium as well as atrophy of prostaic glandular tissue were observed in males exposed to 1000 ppm.

-      28-day rat: spermatogenesis in the testes and the number of spermatozoa in the epididymis were found to be reduced at 600 mg/kg bw/d (Fankhauser 1992).

A GLP compliant study was performed in accordance with OECD 416. Ten weeks after initiation of exposure to the test material at dietary levels of 0, 20, 200 or 2000 ppm the Tif:RAIf (SPF) rats (30 animals per sex and dose level) were paired. Parents were mated 1:1 until positive mating occurred or for 19 days, whichever came first. After weaning and a premating period of 10 weeks, F1 animals were mated to produce the F2 generation. The animals were continuously exposed to the test substance admixed to feed in two successive generations (F0 and F1). Dams were allowed to litter and suckle naturally. Litters were culled to 4 male and 4 female pups, where possible, on day 4 post partum. Clinical signs, body weights, feed consumption, mating parameters, gestation and delivery parameters, pup survival, and physical and behavioural development (surface righting and eye opening) were recorded. A gross necropsy examination was performed on all pups not selected for mating. All parental animals were necropsied after weaning of their offspring and subjected to pathological examination. Histopathology was performed on the sexual organs and the apparent target organs liver and spleen (as identified by organ weight changes). It is concluded that reproductive parameters including gonadal function, mating behaviour, conception, parturition, lactation and weaning, as well as sex organ histopathology were not affected in this study. The dose of 200 ppm was a NOAEL for parents and offspring. At 2000 ppm, parental and offspring body weights were reduced, and eye opening was slightly delayed in pups, and histopathology of adults revealed changes in liver, spleen, and the pituitary. At 200 ppm, a marginal increased incidence of minimal liver hypertrophy was observed in few parental males (17 %), but this finding was not correlated with any liver weight increase at this dose level and therefore not considered as toxicological relevant. 

The classification of the test substance as Repr. 2; H361fd, “Suspected of damaging fertility and the unborn child” is adopted.

Effects on developmental toxicity

Description of key information

- Oral: NOAEL = 10 mg/kg bw/day (maternal toxicity and developmental toxicity), no evidence of teratogenicity, rabbits, gavage, OECD 414, FitzGerald 1992a

- Oral: NOAEL = 30 mg/kg bw/day (maternal toxicity and developmental toxicity), no evidence of teratogenicity, rats, gavage, OECD 414, FitzGerald 1992b

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Sep 1991 to 15 Oct 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA FIFRA 83-3 "Teratogenicity study"

Version / remarks:

October 1982

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA TSCA 798.4900 "Developmental toxicity study",

Version / remarks:

August 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF 59 NohSan No. 4200, "Teratogenicity study"

Version / remarks:

January 1985

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

other: Thomae Russian breed, Chbb:HM

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: at least 3 months
- Weight at study initiation: 2129 to 2661 g
- Housing: The study was carried out under optimal hygienic condition (OHC). The animals were housed individually in Heinkel batteries with wire grid floor.
- Diet: ad libitum, Pelleted, certified standard diet
- Water: tap water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 16
- Photoperiod (hrs dark / hrs light): 12/12 (6 a.m. to 6 p.m.)

IN-LIFE DATES:
16 Sep 1991 to 15 Oct 1991

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% (w/w) aqueous solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance-vehicle mixtures were prepared fresh weekly with a high-speed homogenizer. During administration the homogeneity of the mixtures was maintained by means of a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet: weekly

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of approximately 2 mL volume were taken once before dosing and once after dosing. The samples from before dosing were taken from the top, middle and bottom of the container; the samples from after dosing were taken from the middle of the container.
All samples were stored at approximately -20 degrees Celsius until analysis. The samples for the stability test were allowed to stand at room temperature for the duration of the dosing period and then frozen.

Details on mating procedure:

IMPREGNATION PROCEDURE: ARTIFICIAL INSEMINATION
Nulliparous females, individually identified by an ear tag, were quarantined and acclimated to the facility environment and diet for at least seven days before being inseminated and placed on study. During this time, they were checked for general health. Only healthy animals were placed on study. Approximately 1 hour after i.v. injection of a synthetic releasing hormone (0.1 - 0.2 mL/kg Receptal (busereline acetate)), females were artificially inseminated with diluted semen (in sterile physiological saline) from bucks of the same strain.
The day of insemination was designated Day 0 of pregnancy.
Note: With insemination by natural mating, day of pregnancy is also referred to as day post coitum (p.c.) in raw data and report. In the present study using artificial insemination, the term "post coitum" or "p.c." is to be read as "post insemination".

Duration of treatment / exposure:

Day 7 through 19 p.c.

Frequency of treatment:

Daily

Duration of test:

13 days

Dose / conc.:

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

Remarks:

Group 2, low dose.

Dose / conc.:

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

Remarks:

Group 3, mid dose.

Dose / conc.:

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

Remarks:

Group 4, high dose.

No. of animals per sex per dose:

20

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous range-finding study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, including mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Time schedule for examinations: days 4, 7, 12, 16, 20, 24 and 29 p.c.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: 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 (including contents)
- Number of corpora lutea: Yes (in each ovary)
- Number of implantations: Yes (life and dead)
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: abortion sites

Fetal examinations:

- External examinations: Yes: all per litter (All body regions (e.g. generalized or localized edema, hemorrhage), extremities (e.g. absent, shortened, deformed, additional), tail (e.g. absent, shortened, kinked), trunk (e.g. failure of closure of spinal cord, atresia of a body orifice, omphalocele), head (e.g. absent, reduced, deformed), brain (e.g. cranioschisis, encephalocele, exencephaly), eyes (e.g. absent, reduced, open), pinnae (e.g. absent), jaws (e.g. absent, shortened), oral orifice (e.g. absent), cleft lip/cleft palate).
- Soft tissue examinations: Yes: all per litter (skin, skull elements, central nervous system, eyes, body cavities, respiratory system, digestive system, endocrine system, circulatory system, excretory system, genital system).
- Skeletal examinations: Yes: half litter (cranial skeleton, axial skeleton, appendicular skeleton). Additionally half litter is subjected to examination of the skull for wide fontanels of the fronto-parietal region of the skull.

Statistics:

Statistical analysis of continuous data (e.g. body weight, feed consumption) was performed using the Analysis of Variance Procedure (ANOVA) followed by Dunnett's t-Test in case of a significant result in the ANOVA.
Categorical data (e.g. malformation counts) were analysed using Chi-Square test followed by Fisher's Exact test in case of a significant result in the Chi-Square test.
Non-parametric data (e.g. mean percent affected foetuses/litter) were analysed using the Kruskal-Wallis nonparametric analysis of variance test followed by Mann-Whitney u-test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One dam in the 75 mg/kg group had hair loss from day 26. This was not considered to be treatment-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Three animals died prematurely: one dam in the control group on day 16, and two in the 125 mg/kg group on days 16 and 17 respectively. One of these had haemorrhagic perineal discharge on the previous day 15.
Another dam in the 125 mg/kg group was electively sacrificed after abortion on day 19.
Although there were no necropsy findings to differentiate the death of the control dam and the two in the 125 mg/kg group, the two deaths and the abortion in the 125 mg/kg group were presumed to be treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights were dose-relatedly reduced in the 75 and 125 mg/kg groups, compared to controls, significantly in the 125 mg/kg group from day 18 on.
At 75 mg/kg bw/d, mean maternal body weight gain was reduced from days 7 to 19, achieving 38% of the control value. At 125 mg/kg bw/d, there was even a loss of mean body weight, particularly from days 16 to 20. A statistically significant difference of mean body weights from control was achieved from day 18.
Body weights and body weight gain were unaffected by treatment in the 10 mg/kg group.
Mean body weight and body weight gain values can be found in table 1 in 'Any other information on results incl. tables'.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption was significantly and dose-dependently reduced in the 75 and 125 mg/kg groups during the treatment period (days 7 to 19 of pregnancy).
Feed consumption in the 10 mg/kg group was not affected.

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

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:

effects observed, non-treatment-related

Description (incidence and severity):

In the control group, the dam which was found death had haemorrhagic contents of the abdominal cavity and gall bladder dilatation.
In the 10 mg/kg group, one dam had fluid stomach contents and gall bladder dilatation. Another dam had a constriction in the middle of both uterine horns (and was not pregnant).
In the 75 mg/kg group, one dam had a dilated gall bladder.
In the 125 mg/kg group, the dam killed after aborting on day 19 and the dam which was found dead on day 16 had gas in stomach and gall bladder dilatation. Another dam, found dead on day 17 had mottled dark red lungs.
All the above findings, with the exception of abortion, are commonly observed at necropsy, and the findings in the high dose animals can therefore not be attributed with any certainty to the treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Mean carcass weights were slightly lower than controls in the 75 and 125 mg/kg groups, and net weight loss slightly higher, but these effects were not statistically significant.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

One dam in the 125 mg/kg group had an abortion on day 19.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Preimplantation losses did not differ between groups.
Post-implantation losses as a whole were dose-relatedly increased in the 75 and 125 mg/kg groups.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

One dam in the 75 mg/kg group, and three in the 125 mg/kg group had total resorptions.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Early resorptions (mean percent per animal) were dose-relatedly increased in the 75 and 125 mg/kg groups.
Late resorptions were not significantly increased in any treatment group or the control group.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There were no early deliveries.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

The number of pregnant animals per group were: 17/20, 17/20, 18/20 and 19/20 for the control to high dose group, respectively.
The number of dams per group with viable fetuses at scheduled necropsy was 16, 17, 17, and 13 for control to high dose group, respectively.

Other effects:

no effects observed

Description (incidence and severity):

Gravid uterus weights were not significantly affected by treatment.

Details on maternal toxic effects:

An overview of the number of pregnant dams and dams with; live foetuses, total resorption, early delivery or abortion can be found in table 2 in 'any other information on results incl tables.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights were not significantly affected by treatment.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

The number of live offspring was reduced in the 125 mg/kg group. The number of live offspring was; 103, 116, 110 and 72 for the control to high dose group, respectively.
It should be noted that in none of the dams dead foetuses were found.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Fetal body weight was not significantly affected by the treatment.
Mean litter size was lower in the 125 mg/kg group than in controls.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One foetus in the 10 mg/kg group had a domed head (and was found to have hydrocephalus at visceral examination). This finding has been observed in historical controls, and is not considered treatment-related.
An overview of the external malformations can be found in table 4 in 'Any other information on results incl. tables'.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

According to the authors there were no skeletal malformations in this study. However, reduced pubis was observed in the 75 and 125 mg/kg groups and reported as anomaly. This finding is considered to be a malformation according to the website “www.devtox.org”, as reported in the 'Renewal Assessment Report' on the substance.
An overview of the skeletal malformations can be found in Table 5 in 'Any other information on results incl. tables'.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations were seen in one 10 mg/kg litter (hydrocephalus), in one 75 mg/kg litter (unilateral renal and ureteral aplasia), and in one 125 mg/kg litter (unilateral renal and bilateral ureteral aplasia).
Two anomalies were seen: small gall bladder in one 10 mg/kg foetus, and small liver in one 125 mg/kg foetus.
Because of their low incidence, lack of dose-relationship, and occurrence in historical control data, these findings were not regarded as treatment related. There were no other remarkable visceral observations in the study.
An overview of the visceral malformations can be found in table 4 in 'Any other information on results incl. tables'.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

EXTERNAL ANOMALIES:
The foetal incidence of position anomaly of one or both forelimbs was dose-relatedly increased, and was significantly higher than controls in the 125 mg/kg group. This was considered to be a treatment effect. The finding consists of flexure of the forepaw at the wrist; it is most likely due to restriction of movement in the uterus; in absence of related morphological findings, it is not categorised as a malformation.
An overview of the external anomalies can be found in table 4 in 'Any other information on results incl. tables'.

SKELETAL ANOMALIES:
- In the 125 mg/kg group, there was an increased incidence of fused sternebrae (2-3, 3-4, 4-5).
- In the 125 and 75 mg/kg groups, there was a dose-related increase in reduced pubis.

SKELETAL VARIATIONS:
Variations occurred in almost all litters. The most frequent variations seen in this study were poor or absent ossification of sternebra-5 and caudal vertebral centers. The following were regarded as treatment-related:
- in the 125 mg/kg group, an increased incidence of poor ossification of metacarpal-1, talus, and medial phalanx of anterior digit-5, and additional caudal vertebral centers.
- in the 125 and 75 mg/kg groups, a dose-related increase in incidence of additional 13th rib(s).
An overview of skeletal anomalies and variations can be found in Table 5 in 'Any other information on results incl. tables'.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reduction in number of live offspring

external malformations

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

skeletal: pelvic girdle

Description (incidence and severity):

No indication of severity givin by the authors

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

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:

yes

ANALYTICAL VERIFICATION OF DOSES:

The results obtained for the content, homogeneity and stability of the test substance in bidistilled water with 0.5% CMC are summarized below:

- The mean concentrations of the homogeneity samples were found to be in the range from 82.4% to 101.6% of the nominal concentrations.

- The homogeneity varied in the range from -2% to +4% of the mean concentrations.

- The test substance was found to be stable in bidistilled water with 0.5% CMC at room  temperature for the period of dosing.

Table 1. calculation of corrected body weight gain of dams (day 6 to day 21 p.c.)

Parameter / dose	0 mg/kg	10 mg/kg	75 mg/kg	125 mg/kg
Number of dams	16	17	17	13
Body weight (g); day 0	2382 d	2358	2373	2355
Body weight (g); day 29	2633 d	2647	2562	2501 *
Body weight gain (g), day 7 to 19	87 d	92	33 *	-29 **
Body weight gain (g), day 0 to 29	251 d	289	189	146
Mean gravid uterus weight [g]	348	345	334	282
Mean carcass weight [g]	2284	2302	2228	2219

d = ANOVA; Dunnet-Test: 5 % (*) or 1 % (**) level

Table 2. fertility data

Parameter / dose	0 mg/kg	10 mg/kg	75 mg/kg	125 mg/kg
Females assigned	20	20	20	20
Dams pregnant	16	17	18	16
Dams with live fetuses on day 29	16	17	17	13
Dams with total resorptions	0	0	1	3
Dams delivering early	0	0	0	0
Dams aborting	0	0	0	1

Table 3. Caesarean section data

	Group	Group 1	Group 2	Group 3	Group 4
	Group name	Control	Low dose	Mid dose	High dose
	Dose level	0 mg/kg	10 mg/kg	75 mg/kg	125 mg/kg
Pregnant, used for calculation	n	16	17	18	16
No. of corpea lutea per animal	mean	8.3	9.0	8.8	7.8
No. of implantation sites per animal	mean	6.8	7.2	6.9	6.3
% of preimplantation loss per animal	mean	20.3	20.5	22.0	20.5
No. of foetuses per animal	mean	6.4	6.8	6.1	4.5
-% of which alive		100	100	100	100
-% of which dead		0	0	0	0
No. of early resorptions per animal	mean	0.3	0.4	0.8	1.8
No. of early resorption as % of implantation per animal	mean%	3.1	5.6	12.4	26.3
No. of early+late resorptions per animal	mean	0.3	0.4	0.8	1.8
No. of early+late resorption as % of implantation per animal	mean%	7.0	9.6	24.6	42.7
No. of postimplantation loss per animal	mean	0.3	0.4	0.8	1.8
No. of postimplantation loss as % of implantation per animal	mean%	7.0	9.6	24.6	42.7

Table 4. fetal external and visceral data 

Foetal Data - External and Visceral Examination (No. of foetuses / No. of litters affected)
Observation	0 mg/kg	10 mg/kg	75 mg/kg	125 mg/kg
Foetuses evaluated / Litters evaluated	103 / 16	116 / 17	110 / 17	72 / 13
Total external abnormalities	0 / 0	1 / 1	4 / 2	6** / 4
- position anomaly forelimb (A, 1)	0 / 0	1 / 1	4#/ 2	6**#/ 4
Total visceral malformations/abnormalities	0 / 0	3 / 2	1 / 1	2 / 2
- domed head (A)	0 / 0	1 / 1	0 / 0	0 / 0
- external hydrocephalus (M)	0 / 0	2 / 1	0 / 0	0 / 0
- internal hydrocephalus (M)	0 / 0	1 / 1	0 / 0	0 / 0
- small gall bladder (A)	0 / 0	1 / 1	0 / 0	0 / 0
- small liver (A)	0 / 0	0 / 0	0 / 0	1 / 1
- renal aplasia (M)	0 / 0	0 / 0	1 / 1	1 / 1
- ureter aplasia (M)##	0 / 0	0 / 0	1 / 1	1 / 1

M= malformation, A = Anomaly, 1 = flexure of the forepaw at the wrist,

Chi-square + fisher’s exact: ** = p< 0.01;

# also increased in comparison to historic controls; ## not observed in historic controls

 Table 5. skeletal examination data

Foetal Data - Skeletal Examination (No. of foetuses / No. of litters affected)
Observation	0 mg/kg	10 mg/kg	75 mg/kg	125 mg/kg
Foetuses evaluated / Litters evaluated	103 / 16	116 / 17	110 / 17	72 / 13
Total Malformations	0 / 0	0 / 0	0 / 0	0 / 0
Total Mean Anomalies	11 / 7	23 / 12	15 / 8	28*# / 11
- fused sternebrae 2-3	0 / 0	0 / 0	0 / 0	6**#/ 2
- fused sternebrae 3-4	5 / 4	6 / 3	3 / 3	18**#/ 8
- fused sternebrae 4-5	2 / 2	7 / 6	4 / 3	19**#/ 8
- reduced pubis###	0 / 0	0 / 0	2##/ 2	3##/ 2
Total Variations	80 / 16	95 / 17	90 / 17	67 / 13
- additional caudal vertebral centers	14 / 10	17 / 10	21 / 13	31**/10
- additional rib 13	2 / 2	2 / 2	11*/ 6	21**#/ 7
- poor ossification of metacarpal-1 (1	1 / 1	2 / 2	2 / 2	10**/ 5
- poor ossification of talus (1	1 / 1	2 / 1	3 / 2	8**/ 4
- poor ossif. of medial phalanx of anterior digit-5 (1	9 / 6	19 / 11	14 / 9	22**/ 9

(1 = common variation normally indicating a slight delay in foetal development / ossification chi-square + fisher’s exact: ** = p< 0.01, * = p< 0.05;

# also increased in comparison to historic controls, ## not observed in historic controls

###Reduced pubis was reported as an anomaly in the report, but is considered to be a malformation according to the 'Renewal Assessment Report' on the substance

Conclusions:

- Maternal toxicity (reduced feed consumption and body weight gain; two deaths at 125 mg/kg) and increased post-implantation losses and fetal anomalies and variations consistent with delayed development were seen in the 75 and 125 mg/kg groups.
- The No Observed Adverse Effect Level (NOAEL) for rabbit dams and foetuses in this study was 10 mg/kg.
- There was no evidence for teratogenicity.

Executive summary:

In a GLP compliant teratogenicity study, performed in accordance with OECD 414, the test substance was tested for its embryonic, foetotoxic, and teratogenic potential in rabbits. The test material was administered by gavage in an aqueous solution of carboxymethylcellulose (0.5% w/w) at daily doses of 0, 10, 75 and 125 mg/kg body weight to 20 inseminated Russian Chbb:HM rabbits per group from day 7 to 19 of pregnancy inclusive, using a dose volume of 4 mL/kg body weight. Dams were killed on day 29 of pregnancy, just prior to the expected delivery and foetuses were removed by Caesarean section for subsequent external, visceral and skeletal examination.

One dam in the control group died on day 16. Two dams in the 125 mg/kg group died on day 16 and 17 respectively. Another dam in the highest treatment group was sacrificed after abortion on day 19. The deaths in the 125 mg/kg group were presumed to be treatment-related. Mean body weights were dose-relatedly reduced in the 75 and 125 mg/kg groups, compared to controls. At 75 mg/kg, mean maternal body weight gain was reduced from days 7 to 19, achieving 38% of the control value. At 125 mg/kg, there was even a loss of mean body weight, particularly from days 16 to 20. Body weights and body weight gain were unaffected by treatment in the 10 mg/kg group. Feed consumption was significantly and dose-dependently reduced in the 75 and 125 mg/kg groups during the treatment period (days 7 to 19 of pregnancy). Gross pathological examination of the dams did not reveal findings which are uncommonly observed at necropsy. The findings in the high dose animals can therefore not be attributed with any certainty to the treatment.

Preimplantation losses did not differ between groups. Post-implantation losses were dose-dependently increased in the 75 and 125 mg/kg groups and were based on early resorptions. Additionally, at the highest dosage group, one abortion and three completely resorbed litters were recorded. Mean litter size was reduced at 125 mg/kg bw/day. Foetal body weights were not affected. The total number of external abnormalities is significantly higher in the group receiving the highest dosage (6/72 foetuses compared with 0/103 foetuses in the control group). The foetal incidence of forelimb position anomalies was increased in the 125 mg/kg group. There were no treatment-related visceral or skeletal malformations. Incidence of the following skeletal findings was increased in the 125 mg/kg group: fused sternebrae (anomaly), reduced pubis (reported by the authors as anomaly, but considered a malformation by the 'Renewal Assessment Report' on the substance), 13thribs (variations), poor ossification of metacarpal-1, talus, and medial phalanx of anterior digit-5 (variations). In the 75 mg/kg the following skeletal finding were observed: reduced pubis (reported by the authors as anomaly, but considered a malformation by the 'Renewal Assessment Report' on the substance) and 13thribs (variation). Maternal toxicity (reduced feed consumption and body weight gain; two deaths at 125 mg/kg) and increased post-implantation losses and foetal anomalies and variations consistent with delayed development were seen in the 75 and 125 mg/kg groups. The No Observed Effect Level (NOAEL) for rabbit dams and foetuses in this study was 10 mg/kg. There was no evidence for teratogenicity.

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 Juny 1991 to 20 Aug 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA FIFRA 83-3 "Teratogenicity study"

Version / remarks:

October 1982

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA TSCA 798.4900 "Developmental toxicity study"

Version / remarks:

August 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF 59 NohSan No. 4200, "Teratogenicity Study"

Version / remarks:

January 1985

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Tif: RAIf (SPF)

Remarks:

hybrids of RII/1 x RII/2

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 8 weeks on day 0 p.c.
- Weight at study initiation: 178 to 222 g on day 0 p.c.
- Housing: The experiment was carried out under optimal hygienic condition (OHC). The animals were housed individually in Macrolon cages with wire mesh tops and with standardized granulated soft wood serving as bedding material.
- Diet: ad libitum, Pelleted, certified standard diet
- Water: tap water ad libitum
- Acclimation period: at least 7 days between delivery from the production facility and the first treatment of day 6 p.c.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): ~16
- Photoperiod (hrs dark / hrs light): 12/12 (6 a.m. to 6 p.m.)

IN-LIFE DATES:
30 July 1991 to 20 Aug 1991

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance-vehicle mixtures were prepared in a glass-bead mill. During administration the homogeneity of the mixtures was maintained by means of a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet: weekly

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg actual daily body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of approximately 2 mL volume were taken once before dosing and once after dosing. The samples from before dosing were taken from the top, middle and bottom of the container; the samples from after dosing were taken from the middle of the container.
All samples were stored at approximately -20 degrees Celsius until analysis. The samples for the stability test were allowed to stand at room temperature for the duration of the dosing period and then frozen.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/3
- Length of cohabitation: Pregnant females were removed and the procedure repeated for remaining females until sufficient dams were produced. Start of cohabitation 30 July, end of cohabitation 7 August.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 through 15 post coitum

Frequency of treatment:

Daily

Duration of test:

10 days

Dose / conc.:

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

Remarks:

Group 2, low dose.

Dose / conc.:

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

Remarks:

Group 3, mid dose.

Dose / conc.:

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

Remarks:

Group 4, high dose.

No. of animals per sex per dose:

24

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous range-finding study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, including mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Time schedule for examinations: days 6, 11, 16 and 21 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: 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: Yes
- Number of implantations: Yes (life and dead)
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: abortion sites and uterine horns showing no implantation sites were placed into an aqueous solution of ammonium sulphide to visualize possible haemorrhagic alterations of such sites, according to the method of Salewski (1964).

Fetal examinations:

Foetuses were assigned to either visceral or skeletal evaluation at an approximate 1:1 ratio in each litter, independent of sex (starting with skeletal). In the case of gross anomaly and/or malformation, however, foetuses were allocated to one of the two techniques depending on the type and incidence of the finding.
- External examinations: Yes: all per litter ((all body regions (e.g. generalised or localised oedema, haemorrhage), extremities (e.g. absent, shortened, deformed, additional), tail (e.g. absent, shortened, kinked), trunk (e.g. failure of closure of spinal cord, atresia of a body orifice, omphalocele), head (e.g. absent, reduced, deformed), brain (e.g. cranioschisis, encephalocele, exencephaly), eye (e.g. absent, reduced, open), pinnae (e.g. absent), jaws (e.g. absent, shortened), oral orifice (e.g. absent), cleft lip/cleft palate)
- Soft tissue examinations: Yes: half per litter
Skin
Central nervous system – brain (olfactory bulbs, cerebrum lateral and medial ventricles), spinal cord
Eyes – lens, vitreous, retina
Body cavities – thorax and abdomen, including diaphragm
Respiratory system – nasal cavity (nasal septum, turbinates, choanae), trachea, bronchi, lungs, pleura
Digestive system – oral cavity, palate, tongue, oesophagus, stomach, intestine, rectum, liver, peritoneum
Endocrine system – thyroid, pancreas, adrenals, thymus
Circulatory system – spleen, pericardium, heart (atria, ventricles septae), major vessels
Excretory system – kidney (renal papillae, renal pelvis), ureters, urinary bladder
Genital system – testes, epididymides, vas deferens, seminal vesicles; ovaries, oviduct, uterus
- Skeletal examinations: Yes: half per litter
Cranial Skeleton – nasal, premaxillary, maxillary, zygomatic, frontal and parietal bones; fontanel; interparietal, occipital and exoccipital bones; mandibula
Axial Skeleton – cervical, thoracic, lumbar and sacral vertebrae and centers; ribs, sternebrae
Appendicular Skeleton – shoulder girdle (clavicle, scapula), forelimbs (humerus, ulna, radius, metacarpals, proximal phalanges, distal phalanges), pelvic girdle (ilium, ischium, pubis), hindlimbs (femur, tibia, fibula, calcaneus, metatarsals, proximal phalanges, distal phalanges).

Statistics:

Statistical analysis of continuous data (e.g. body weight, feed consumption) was performed using the Analysis of Variance Procedure (ANOVA) followed by Dunnett's t-Test in case of a significant result in the ANOVA.
Categorical data (e.g. malformation counts) were analysed using Chi-Square test followed by Fisher's Exact test in case of a significant result in the Chi-Square test.
Non-parametric data (e.g. mean percent affected foetuses/litter) were analysed using the Kruskal-Wallis nonparametric analysis of variance test followed by Mann-Whitney u-test.

Historical control data:

Historical control data available on fetal external observations, fetal visceral observations, fetal skeletal malformations, fetal skeletal anomalies and fetal skeletal variations.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One dam in the 100 mg/kg group and one in the 300 mg/kg group had hair loss starting on day 15 and 16 p.c. respectively, and one dam in the 300 mg/kg group had a neck wound on day 15 p.c. and then neck crust/scurf until necropsy.
These findings were considered to be not test substance related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights were lower in the 300 mg/kg group than controls during and after treatment, but there were no statistically significant differences to controls. A statistical significant reduction in body weight gain was observed in animals receiving a dose of 300 mg/kg from day 6 to 11.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption was significantly and dose-dependently reduced in the intermediate (100 mg/kg) and high dose (300 mg/kg) groups during the treatment period (days 6 to 15 p.c.). Feed consumption in the low dose group (30 mg/kg) was not affected.

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Gravid uterus weights were not affected by treatment. There was a dose-related reduction in mean carcass weight; net body weight was significantly and dose-relatedly reduced in animals receiving 100 and 300 mg/kg test substance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

One dam in the intermediate dose group (100 mg/kg) had an abnormally positioned uterus (the left uterine horn lay dorsal to the colon). This finding was not related to the treatment and had no effect on pregnancy or delivery.
There were no other notable observations at maternal necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

There were no abortions in any treatment group or control group.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Overall pre- and post-implantation losse were not affected.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

One dam receiving 300 mg/kg had total resorptions.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Foetal weights were not affected.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Number of live foetuses per litter was not affected.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Number of live fetuses per litter and fetal weights were not affected by treatment.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- The following external malformations were seen in the 100 mg/kg group in four foetuses from three litters: generalized oedema, rachischisis, and omphalocele.
- In the 300 mg/kg group, one foetus had an omphalocele. External anomalies were seen in three foetuses and litters in the 100 mg/kg group: tail hypoplasia, kinked tail, brachycaudia and right hindlimb position anomaly.
- In the 300 mg/kg group, one foetus had position anomaly of both hindlimbs ("Position anomaly" of hindlimb is defined as the limb resting with the foot on or over the saggital midline of the body. It may arise from restriction of movement in the uterus; in the absence of related morphological findings, it is not categorised as a malformation.)
- Because of their scattered occurrence and lack of dose-relationship, and most have occurred in historical controls, it is concluded that these findings were not treatment-related.
- An overview of the external examination data can be found in Table 2 in 'Any other information on results incl. tables'.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Incidence of the following skeletal findings was increased at 300 mg/kg bw/day. The effect was statistical significant only with regard to the number of foetuses:
- Displaced pubic bone(s); A defect of this type is not mentioned in the list of defects occurring in historic controls and is therefore tentatively categorized as a malformation of minor severity. It is not regarded as indicating teratogenicity of the
test substance because of its low frequency of occurrence and lack of severity.
- One foetus in the 100 mg/kg group had all sacral vertebrae missing. This isolated finding was not regarded as treatment-related.

Visceral malformations:

no effects observed

Description (incidence and severity):

- Visceral malformations were seen in one control foetus and one foetus from the 100 mg/kg group: hypoplasia of the left cardiac ventricle, abnormal pulmonary lobulation (no lobulation on the right lung, but normal size), and cystic liver and kidney. This foetus had generalised oedema at external examination.
- Visceral anomalies were seen in one foetus from the 30 mg/kg group (ectopic left kidney) and one foetus from the 100 mg/kg group (unilateral renal pelvic dilation; this foetus had omphalocele, brachycaudia, and a unilateral hindlimb position anomaly at external examination). Variations, consisting of enlarged thymus or accessory lobulets on the liver, were seen in 6, 1, 3, and 2 foetuses and litters from the control to high dose group, respectively.
- None of the above findings was considered to be treatment-related.
- An overview of the visceral examination data can be found in Table 2 in 'Any other information on results incl. tables'.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

SKELETAL ANOMALIES AND VARIATIONS
Incidence of the following skeletal findings was increased at 300 mg/kg bw/day. The effect was statistical significant only with regard to the number of foetuses:
- asymmetrically shaped sternebra-5
- thickening of ischium (anomaly)
- shortened rib 13, and absent ossification of metatarsal-1 and of proximal phalanges of anterior digit 5 and posterior digits 2 to 5 (variation)
- dumbbell-shaped thoracal vertebral centers, this variation lay in the range of historical control values (variation)
Incidence of the following skeletal findings was increased at 100 mg/kg bw/day. The effect was statistical significant only with regard to the number of foetuses:
- dumbbell-shaped cervical vertebral centers (variation)

The significantly increased foetal incidence of dumbbell-shaped thoracic vertebral centers in the 300 mg/kg group was not regarded as treatment-related, since it lay within the range of historical control values (foetal incidence 3.8%, litter incidence 27.3%; range of historical control values 0.6-4.5% and 4.5-30%, respectively).
The significantly increased foetal incidence of poor ossification of proximal phalanges of posterior digit 5 in all treated groups compared to controls was not regarded as treatment-related, because the control incidences in this study were lower than normal (foetal incidence 2.5%, litter incidence 17.4%; range of historical control values 3.1-14.8% and 22.7-70.0%, respectively), the incidences in the treated groups were all within the range of historical control values, and the apparently increased incidences relative to controls were not dose-related.
All treated groups had higher foetal incidence of bipartite cervical vertebral centers than controls (litter incidences were not significantly affected). Incidences were within historical control limits in all groups, and there was no indication of a dose-relationship. This effect was therefore considered not to be treatment-related.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

not specified

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: pelvic girdle

Description (incidence and severity):

Displaced pubic bones; malformation of minor severity.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

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:

yes

ANALYTICAL VERIFICATION OF DOSES:

The results obtained for the content, homogeneity and stability of the test substance in bidistilled water with 0.5% CMC are summarized below:

- The mean concentrations of the homogeneity samples were found to be in the range from 92.2% to 104.6% of the nominal concentrations.

- The homogeneity varied in the range from -5% to +4% of the mean concentrations.

- The test substance was found to be stable in bidistilled water with 0.5% CMC at room  temperature for the period of dosing.

Table 1. fertility data and Caesarean section data

Parameter / dose	0 mg/kg	30 mg/kg	100 mg/kg	300 mg/kg
Females assigned Dams pregnant	24 23	24 22	24 23	24 23
Dams with live fetuses on day 21	23	22	23	22
Dams with total resorptions	0	0	0	1
Dams delivering early	0	0	0	0
Dams aborting	0	0	0	0
Corpora lutea / dam (mean)	17.0	17.0	16.6	15.9
Implants / dam (mean)	13.6	14.0	14.0	14.1
Preimplantation loss [%]	19.6	16.9	14.9	11.6
Postimplantation loss [%]	2.4	6.1	3.8	6.7
Live fetuses / Dam (mean)	13.2	13.2	13.5	13.0
Dead fetuses	0	0	0	0
Resorptions/Dam - Early (mean)	0.3	0.9	0.5	0.9
Resorptions/Dam - Late (mean)	0	0	0	0
Pup body weight [g] (mean)	5.6	5.5	5.6	5.4

 Table 2. External and visceral examination data

Foetal Data - External and Visceral Examination (No. of foetuses / No. of litters affected)
Observation / dose	0 mg/kg	30 mg/kg	100 mg/kg	300 mg/kg
Total foetuses / litters affected	1 / 1	1 / 1	6 / 3	2 / 2
- Omphalocele (M)	0 / 0	0 / 0	2 / 1	1 / 1
- Generalised edema (M)	0 / 0	0 / 0	2 / 2	0 / 0
- Rachischisis (M)	0 / 0	0 / 0	1 / 1	0 / 0
- Deformed brain (M)	1 / 1	0 / 0	0 / 0	0 / 0
- Position anomaly hindlimb (A)	0 / 0	0 / 0	1 / 1	1 / 1
- Tail hypoplasia (A)	0 / 0	0 / 0	1 / 1	0 / 0
- Kinked tail (A)	0 / 0	0 / 0	1 / 1	0 / 0
- See (1) below	0 / 0	0 / 0	1 / 1	0 / 0
- Ectopic left kidney (A)	0 / 0	1 / 1	0 / 0	0 / 0
- See (2) below	0 / 0	0 / 0	1 / 1	0 / 0

M = malformation, A = anomaly

(1) = hypoplasia of l. cardiac ventricle (M), abnormal pulmonary lobulation (M), cystic liver and kidney (M); this fetus had generalised edema at external examination

(2) = renal pelvic dilation (A), brachycaudia (A); this foetus had omphalocele, and unilateral hindlimb position anomaly at external examination

 Table 3. Skeletal examination data

Foetal Data - Skeletal Examination (No. of foetuses / No. of litters affected)
Observation / dose	0 mg/kg	30 mg/kg	100 mg/kg	300 mg/kg
No. of foetuses / litter evaluated	158 / 23	150 / 22	163 / 23	157 / 22
Total skeletal malformations	0 / 0	0 / 0	1 / 0	4**/ 3
- Displaced pubic bones (1	0 / 0	0 / 0	0 / 0	4**##/ 3
Total skeletal anomalies	3 / 3	4 / 4	5 / 5	17**/ 12
- Thickened ischium of pelvis	0 / 0	0 / 0	0 / 0	2##/ 2 (2
- Asymmetrically shaped sternebra-5	1 / 1	0 / 0	0 / 0	5#/ 5
Total Skeletal variations	164 / 23	151 / 22	166 / 23	159 / 22
- Shortened rib 13 (3	13 / 10	11 / 8	16 / 11	40#/ 17
- Absent ossification of metatarsal-1(3	4 / 3	6 / 5	3 / 3	26** / 8
- Absent ossification of prox. phalanges ant.Digit-5 (3	2 / 2	2 / 2	4 / 3	16** / 7
- Absent ossification of post. digit-2 (3	17 / 9	9 / 6	20 / 10	45** / 13
- Absent ossification of post. digit-3 (3	4 / 4	3 / 3	9 / 7	29** / 9
- Absent ossification of post. digit-4 (3	5 / 4	5 / 3	12 / 9	25** / 9
- Absent ossification of post. digit-5 (3	36 / 17	29 / 12	51 / 18	74** / 18
- Dumbbell-shaped thor. vertebral centers (4	0 / 0	1 / 1	3 / 3	6*/ 6
- Dumbbell-shaped cerv. vertebral centers (3	6 / 4	2 / 2	17*#/ 12	6#/ 5
- Bipartite cerv. vert. centres (4	8 / 7	19*/ 11	28**/ 14	23**/ 12

chi-square + fisher’s exact: 5 % (*) or 1 % (**) level;

# also increased in comparison to historic controls; ## not observed in historic controls

1 = rare change with low frequency of occurrence and minor severity

2 = both of these foetuses also had a displaced pubic bone

3 = common variation normally indicating a slight delay in foetal development (ossification)

4 = all incidences within the range of historical control values

Conclusions:

- Maternal toxicity (reduced feed consumption and body weight gain) and foetal skeletal anomalies and variations consistent with delayed ossification were seen in the 100 and 300 mg/kg bw/day groups. A minor skeletal malformation was seen in the 300 mg/kg bw/day group.
- The no observed adverse effect level (NOAEL) for rat dams, embryos and foetuses in the present study was 30 mg/kg bw/day.
- There was considered to be no evidence for teratogenicity.

Executive summary:

In a GLP compliant teratogenicity study, performed in accordance with OECD 414, the test substance was tested for its embryonic, foetotoxic, and teratogenic potential in rats. The test substance was administered by gavage in an aqueous solution of carboxymethylcellulose (0.5% w/w) at daily doses of 0, 30, 100 and, 300 mg/kg body weight to 24 mated Tif:RAIf rats per group from day 6 to 15 of pregnancy inclusive, using a dose volume of 10 mL/kg body weight. Dams were killed on day 21 p.c., just prior to the expected delivery and foetuses were removed by Caesarean section for subsequent external, visceral and skeletal examination. There were no premature mortalities during the study and there were no abortions. Mean maternal body weights were lower in the 300 mg/kg group than controls during and after treatment, but there were no statistically significant differences to controls. Maternal body weight gain was reduced in the 300 mg/kg group from day 6 to 11.Feed consumption was significantly and dose-dependently reduced in the intermediate (100 mg/kg) and high dose (300 mg/kg) groups during the treatment period (days 6 to 15 p.c.). Feed consumption in the low dose group (30 mg/kg) was not affected. Gravid uterus weights were not affected by treatment. There was a dose-related reduction in mean carcass weight; net body weight gain was significantly and dose-relatedly reduced in the 100 and 300 mg/kg groups. Necropsy examination revealed no other significant findings in surviving animals. The number of dams per group with viable foetuses was 23, 22, 23, and 22 in the control to high dose groups, respectively. Preimplantation losses, numbers of implantation sites, and early and late post-implantation losses were comparable between groups. There were no dead or aborted foetuses. The number of live foetuses per litter and foetal weights were not affected by treatment. One dam receiving 300 mg/kg had total resorptions. Changes in litter size and weights and individual foetal body weight were not observed. Furthermore, the number of live foetuses per litter were not affected. There were no foetal external or visceral observations that were considered to be related to treatment. At skeletal examination, four foetuses from three litters in the 300 mg/kg group had displaced pubic bone(s) which is considered to be a malformation. Other finding in the highest treatment group were: asymmetrically shaped sternebra (anomaly), shortened 13^thrib, absent ossification of metatarsal-1 and of proximal phalanges of anterior digit 5 and posterior digits 2 to 5, dumbbell-shaped thoracal vertebral centers, this variation lay in the range of historical control values (variation). In the 100 mg/kg a statistically significant increased incidence of dumbbell-shaped cervical vertebral centres (variation) was found. Maternal toxicity (reduced feed consumption and body weight gain) and foetal skeletal anomalies and variations consistent with delayed ossification were seen in the 100 and 300 mg/kg groups. A minor skeletal malformation was seen in the 300 mg/kg group. The No Observed Adverse Effect Level (NOAEL) for rat dams, embryos and foetuses in the present study was 30 mg/kg body weight/day. There was considered to be no evidence for teratogenicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

GLP compliant OECD 414 studies

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 414 - Rabbit

In a GLP compliant teratogenicity study, performed in accordance with OECD 414, the test substance was tested for its embryonic, foetotoxic, and teratogenic potential in rabbits (FitzGerald 1992a). The test material was administered by gavage in an aqueous solution of carboxymethylcellulose (0.5% w/w) at daily doses of 0, 10, 75 and 125 mg/kg body weight to 20 inseminated Russian Chbb:HM rabbits per group from day 7 to 19 of pregnancy inclusive, using a dose volume of 4 mL/kg body weight. Dams were killed on day 29 of pregnancy, just prior to the expected delivery and foetuses were removed by Caesarean section for subsequent external, visceral and skeletal examination.

One dam in the control group died on day 16. Two dams in the 125 mg/kg group died on day 16 and 17 respectively. Another dam in the highest treatment group was sacrificed after abortion on day 19. The deaths in the 125 mg/kg group were presumed to be treatment-related. Mean body weights were dose-relatedly reduced in the 75 and 125 mg/kg groups, compared to controls. At 75 mg/kg, mean maternal body weight gain was reduced from days 7 to 19, achieving 38% of the control value. At 125 mg/kg, there was even a loss of mean body weight, particularly from days 16 to 20. Body weights and body weight gain were unaffected by treatment in the 10 mg/kg group. Feed consumption was significantly and dose-dependently reduced in the 75 and 125 mg/kg groups during the treatment period (days 7 to 19 of pregnancy). Gross pathological examination of the dams did not reveal findings which are uncommonly observed at necropsy. The findings in the high dose animals can therefore not be attributed with any certainty to the treatment.

Preimplantation losses did not differ between groups. Post-implantation losses were dose-dependently increased in the 75 and 125 mg/kg groups and were based on early resorptions. Additionally, at the highest dosage group, one abortion and tree completely resorbed litters were recorded. Mean litter size was reduced at 125 mg/kg bw/day. Foetal body weights were not affected. The total number of external abnormalities is significantly higher in the group receiving the highest dosage (6/72 foetuses compared with 0/103 foetuses in the control group). The foetal incidence of forelimb position anomalies was increased in the 125 mg/kg group. There were no treatment-related visceral or skeletal malformations. Incidence of the following skeletal findings was increased in the 125 mg/kg group: fused sternebrae (anomaly), reduced pubis both reported as anomaly, but considered a malformation), 13^thribs (variations), poor ossification of metacarpal-1, talus, and medial phalanx of anterior digit-5 (variations). In the 75 mg/kg the following skeletal finding were observed: reduced pubis (reported as anomaly, but considered a malformation) and 13thribs (variation).Maternal toxicity (reduced feed consumption and body weight gain; two deaths at 125 mg/kg) and increased post-implantation losses and foetal anomalies and variations consistent with delayed development were seen in the 75 and 125 mg/kg groups. The no observed effect level (NOAEL) for rabbit dams and foetuses in this study was 10 mg/kg. There was no evidence for teratogenicity.

 

OECD 414 - Rat

In a GLP compliant teratogenicity study, performed in accordance with OECD 414, the test substance was tested for its embryonic, foetotoxic, and teratogenic potential in rats (FitzGerald 1992b). The test substance was administered by gavage in an aqueous solution of carboxymethylcellulose (0.5% w/w) at daily doses of 0, 30, 100 and, 300 mg/kg body weight to 24 mated Tif:RAIf rats per group from day 6 to 15 of pregnancy inclusive, using a dose volume of 10 mL/kg body weight. Dams were killed on day 21 p.c., just prior to the expected delivery and foetuses were removed by Caesarean section for subsequent external, visceral and skeletal examination. There were no premature mortalities during the study and there were no abortions. Mean maternal body weights were lower in the 300 mg/kg group than controls during and after treatment, but there were no statistically significant differences to controls. Maternal body weight gain was reduced in the 300 mg/kg group from day 6 to 11.Feed consumption was significantly and dose-dependently reduced in the intermediate (100 mg/kg) and high dose (300 mg/kg) groups during the treatment period (days 6 to 15 p.c.). Feed consumption in the low dose group (30 mg/kg) was not affected. Gravid uterus weights were not affected by treatment. There was a dose-related reduction in mean carcass weight; net body weight gain was significantly and dose-relatedly reduced in the 100 and 300 mg/kg groups. Necropsy examination revealed no other significant findings in surviving animals. The number of dams per group with viable foetuses was 23, 22, 23, and 22in the control to high dose groups, respectively. Preimplantation losses, numbers of implantation sites, and early and late post-implantation losses were comparable between groups. There were no dead or aborted foetuses. The number of live foetuses per litter and foetal weights were not affected by treatment. One dam receiving 300 mg/kg had total resorptions. Changes in litter size and weights and individual foetal body weight were not observed. Furthermore, the number of live foetuses per litter were not affected. There were no foetal external or visceral observations that were considered to be related to treatment. At skeletal examination, four foetuses from three litters in the 300 mg/kg group had displaced pubic bone(s) which is considered to be a malformation. Other finding in the highest treatment group were: asymmetrically shaped sternebra (anomaly), shortened 13^th rib, absent ossification of metatarsal-1 and of proximal phalanges of anterior digit 5 and posterior digits 2 to 5, dumbbell-shaped thoracal vertebral centers, this variation lay in the range of historical control values (variation). In the 100 mg/kg a statistically significant increased incidence of dumbbell-shaped cervical vertebral centres (variation) was found. Maternal toxicity (reduced feed consumption and body weight gain) and foetal skeletal anomalies and variations consistent with delayed ossification were seen in the 100 and 300 mg/kg groups. A minor skeletal malformation was seen in the 300 mg/kg group. The no observed effect level (NOAEL) for rat dams, embryos and foetuses in the present study was 30 mg/kg body weight/day. There was considered to be no evidence for teratogenicity.

Evaluation of the developmental toxicity studies

Neubert (1992): An opinion to the fetal findings in the rat and rabbit teratogenicity studies was sought from Neubert. He summarised, that all the experimental results known up until now support the case that the effects observed can be regarded as "unspecific", i.e. they occur as a result of general toxic effect to the mother. He mentioned, that these data do not justify to postulate a teratogenic potential for the test substance. With regard to the pelvic lesions he had concluded that it is important to note, that changes in the pelvis, which could not be ascribed to substance-specific changes in the individual studies, occurred in both species. Interestingly, defects of this kind have not been recorded in historic controls in either species.

Re-evaluation (2013): The conclusion of the original review of the document (Neubert, 1992; Neubert, 1992) is supported, i.e., that it is not acceptable and does not contribute any relevant information, that would be supported by experimental data or would explain the occurrence of the findings.

Kaneda and Kodaira (1996): Teratogenicity study in the rats with the test substance: re-examination of skeletal findings (Translated from the Japanese report). Report No. IET 95-0122. Kodaira Laboratories, The Institute of Environmental Toxicology, Suzuki-Cho 2-772, Kodaira-Shi, Tokyo 187, Japan. March 11, 1996. The study was performed according the OECD Guideline 414, adopted May 1981; (equivalent to Directive Directive 87/302/EEC; US EPA Pesticide Assessment Guideline, Subdiv. F. § 83-3, October 1982; Japanese MAFF 59 NohSan No. 4200, January 1985) under GLP conditions. There were no significant deviations from Directive 87/302/EEC. Therefore, the study is considered acceptable. Groups of 15 pregnant female Crj:CD (SD) rats were orally administered the test substance in a 0.5 % aqueous carboxymethylcellulose solution at dose levels of 0, 3 and 30 mg/kg body weight from day 6 to 15 gestation. On day 20 of gestation foetuses were removed by caesarean section. All live foetuses were examined for external alterations. They were then processed for skeletal examination using double staining for cartilage and bone. No treatment-related adverse effects were found in the clinical findings, body weights, body weight gains, food consumption, and gross pathological examination in the dams. The number of live foetuses, percent resorptions, foetal deaths, foetal sex ratio, foetal and placental weights, external findings, and skeletal examination (malformations, variations degree of ossification)revealed no treatment-related adverse effects. Particularly the cartilaginous cervical vertebra bodies were morphologically normal in all specimens. The NOEL was 30 mg/kg, confirming the NOEL determined in the conclusion developmental study on rats.

Re-evaluation (2013): The study (Kaneda and Kodaira, 1996) is considered supplementary (groups consisted of less than 20 pregnant animals. Treatment period was only from day 6 to 15 instead of the period given in the current testing guideline 414. No effects observed in top dose group). It is noted, that another rat strain was tested in this study in another laboratory. Under the conditions of the study, no effects on dams or foetuses were reported up to the highest dose level of 20 mg/kg bw/d.

Fuchs, A. (1997): Review of a teratogenic study in the rat with special regard to skeletal examination. CLE study Nr. 252-239. Covance Lab. Muenster, Germany. The study was performed to provide a re-evaluation of fetal skeletal findings in rat fetuses from the oral teratogenicity study. Since the fetuses were in a somewhat bad condition after having been stored in the archive for approximately five years, the evaluation was reported as sometimes difficult. In cases that it was unclear whether a finding was the result of an artefact or a real abnormality, the finding was recorded as a skeletal observation. This could be a reason, that some abnormalities not observed in the control group of the original study were described in this report (e.g. insertion of pelvic girdle). Some other malformations could not be observed in the five year archived material of the re-evaluation study (e.g. Omphaloceles (umbilical hernia), which occurred only at the highest doses level and Generalised edema). Furthermore, a different terminology was mainly used in this re-evaluation study. But, if the same terms were applied, different incidences were described (e.g. shortened ribs). For these reasons, a comparison of findings in the original report with the re-evaluation results is confined. Therefore, the re-evaluation report is considered not acceptable. The conclusion of this report could not be supported, that the general pattern of observed malformations and variations did not show any treatment-relationship. The re-evaluation has also shown an increased incidence of e.g. rib and pelvic lesions at the highest dose level. 

Re-evaluation (2013): The conclusion of the original review of the document (Fuchs, 1997) is supported, i.e., that the document is not acceptable and does not provide any relevant information, that would explain the occurrence of the findings. Following observation of the study director is emphasised: “Since the fetuses were in a somewhat bad condition after having been stored in the archive for approximately five years, evaluation was sometimes difficult. This was especially true for evaluation of skull bones, the position of ribs and the symmetry of the pelvic and pectoral girdle.” In summary, the specimens were in no condition that would allow a proper re-evaluation of the critical findings described in the original study report.

Fuchs, A. (1997): Review of a teratogenic study in the rabbit with special regard to skeletal examination. CLE study Nr. 252-238. Covance Lab. Muenster, Germany. The study was performed to provide a re-evaluation of fetal skeletal findings in rabbit fetuses from the oral teratogenicity study. Since the fetuses were in a somewhat bad condition after having been stored in the archive for approximately five years, the evaluation was reported as sometimes difficult. This was especially true for the evaluation of pelvic bones, a possible target for developmental effects of pymetrozine. These technical problems could be also a reason, that some abnormalities were observed in the control group of the original study with a lower incidence than in this report (e.g. fused sternebrae). Furthermore, a different terminology was mainly used in this reevaluation study (additional rib 13 vs. extra thoraco-lumbar rib). But, if nearly the same terms were applied, different incidences were described (e.g. fused sternebrae). For these reasons, a comparison of findings in the original report with the re-evaluation results is confined. Therefore, the re-evaluation report is considered not acceptable. The re-evaluation has shown an increased incidence of increased percentage of fetuses with extra thoraco-lumbar ribs at dose levels of 75 and 125 mg/kg bw/d. An increased incidence of pelvic lesions and incomplete ossified pubis’s were observed at all dose levels. But with regard to the technical problems of this re-evaluation, these lesions cannot be unequivocally attributed to the application of the test substance.

Re-evaluation (2013): The conclusion of the original review of the document (Fuchs, 1997) is supported, i.e., that the document is not acceptable and does not provide any relevant information, that would explain the occurrence of the findings. Following observation of the study director is emphasised: “Since the fetuses were in a somewhat bad condition after having been stored in the archive for approximately five years, evaluation was sometimes difficult. This was especially true for evaluation of the pelvic girdle. In 11/103 control fetuses, 28/116 fetuses from group 2, 14/110 fetuses from group 3 and 16/72 fetuses from group 4, the insertion site of the pelvic girdle was not determinable since the hindlimbs, including the bubis and ischium were dislocated or detached.” In summary, the specimens were in no condition that would allow a proper re-evaluation of the critical findings described in the original study report.

In conclusion, the evaluations did not change the outcome of the the developmental studies. 

Justification for classification or non-classification

Based on the available information, the test substance is classified as reprotoxic toxicant (category 2) in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.