Avermectin A1a, 25-cyclohexyl-5-O-demethyl-25-de(1-methylpropyl)-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A two-generation gavage study on rats is available which run to a reliable method under GLP (Fisher, 1991).

The compound administration did not alter the appearance or survival of the dams or sires. No treatment related differences were observed in pregnancy rates, litter size or pup survival. The only treatment related findings were 11% and 17% decrements in pup weight gain during lactation for the F2a and F2b offspring respectively, at the 1.0 mg/kg dose level.

Based on the finding in this 2-generation study with test substance, the No-Observed-Effect-Level (NOEL) is 0.3 mg/kg/day.

 

Series maternal toxicity studies on rats, mice and rabbits are available (Kessedjian, 1988) to find dose ranges for fetotoxicity studies.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Oct 1989 to Aug 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP.

Qualifier:

according to guideline

Guideline:

other: Drug Safety Evaluation Standard Procedures

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charies River Breeding Laboratories, Wilmington, MA.
- Age at study initiation:
- Weight at study initiation: 60 g
- Fasting period before study:
- Housing: individually housed in hanging stainless steel cages with wire mesh fronts and floors
- Diet (e.g. ad libitum): standard ground laboratory diet (Agway Prolab RMH 3200 Meal) ad libitum
- Water (e.g. ad libitum): Drinking water was supplied ad libitum through an automatic watering system (Edstrom Industries) with founts in each cage. Water which was obtained from a municipal water system subject to regulations of the Environmental Protection Agency was passed through a reverse osmosis apparatus.
- Acclimation period: approximately 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70±2
- Humidity (%): 50±5
- Air changes (per hr): approximately 18 times per hour with air filtered through 80-90% efficiency filters and finally through HEPA filters
- Photoperiod (hrs dark / hrs light): illuminated by fluorescent lighting 12 hours a day (7:00 am-7:00pm)

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Solutions of test substance in sesame oil, as prepared for toxicology studies, canbe used for at least one year without refrigerated storage and still have potency maintained.
- Concentration in vehicle: 0.02, 0.06 and 0.12 mg (active moiety) test substance/mL
- Amount of vehicle (if gavage): 0.5 mL per 100 grams of body weight
- Lot/batch no. (if required):
- Purity:

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 5 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After seven days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individual in shoe-box type cages
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Solutions of test substance in sesame oil were submitted for analysis periodically during the study. All samples were within ±15% of their intended concentrations.

Duration of treatment / exposure:

F0 males: approximately 10 weeks;
F0 females: 2 weeks prior to cohabitation and continuously therafter during gestation and lactation;
F1 pups: in utero, and during lactation via sucking and received test substance by gavage from the moment they were weaned and continuously thereafter;
F2a and F2b: in utero, via the milk and by oral gavage

Frequency of treatment:

oral gavage: daily
in utero, via the milk: continuously in feed

Details on study schedule:

- F1 parental animals not mated until 17 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: approximately 28 weeks

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

F0 rats: 45 animals/sex/dose;
F1 pups: 30 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of previous rat studies, dose levels below the 1.5 to 6.0 mg/kg would be needed to aviod potential effects on pup survival. This was confirmed in a toleration trial by traeting pregnant rats with the test substance throughout gestation and lactation, and monitoring pup survival. Pups and dams dosed at 0.1, 0.2 and 0.5 mg/kg seemed unaffected, but at 1.0 mg/kg there was a moderate decrement in pup weight. Based on this information, dose levels of 0.1, 0.3 and 1.0 mg/kg were selected for the present 2-generation study.

Positive control:

None stated

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily on weekdays and at least once daily on weekends and holidays

BODY WEIGHT: Yes
- Time schedule for examinations: weekly prior to cohabiting and during mating period; During gestation, body weight of pregnant F0 females were obtained on days 0,4,7,14 & 21.

FOOD CONSUMPTION:
-Food consumption of males were recorded weekly prior to cohabiting.

OTHER:

Oestrous cyclicity (parental animals):

None stated

Sperm parameters (parental animals):

None stated

Litter observations:

gestation lengths, live and dead pups number for each litter, weight individualiy on day 1,4,7,14, and 21 post-partum.

Postmortem examinations (parental animals):

F0 males were sacrificed after mating and examined macroscopically.
Any F0 females which did not deliver by day 24 of gestation were sacrificed and their uteri and ovaries examined for signs of pregnacy and abnormalities. F0 females were sacrificed after weaning of F1 pups and examined macroscopically for internal and external abnormalities.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21days of age.
- These animals were subjected to postmortem examinations macroscopic examination as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations for abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
Reproductive organs such as vagina, uterus, ovaries, testes, epididymides, seminal vesicles and prostate along with the liver and kidneys were preserved for possible future microscopic examination and weighed liver, kidney and testis, respectively.

Statistics:

None stated

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

High dose level

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

F0 Rats and Their F1 Progeny:
General Observations Pertaining to F0 Animals:
All animals survived the treatment periods (10 weeks for males and 2 weeks for females) prior to mating. Two control animals died during the cohabitation period (Days 64-78): male #15 with no apparent cause of death and female #71 with a ruptured aorta as the apparent cause of death. After the cohabitation period (Day 89) high dose male #284 was found dead and had dark red (mottled) lungs, suggesting that aspiration of dosing solution may have been the initiating factor. High dose female #339 died during parturition.
Collectively, the appearance and behavior of the F0 animals were unaffected by treatment.

Body Weights and Food Consumption of F0 Animals Prior to Mating:
Body weights and food consumption values of the F0 male rats were unaffected by treatment during the approximate 10 week period prior to cohabitation. At week 10 (day 64) weights for the control, low, intermediate and high dose males, respectively were 471.2, 470.3, 467.3 and 480.4 grams. Treatment of the F0 females began 2 weeks prior to mating. Food consumption was not assessed, but body weights during this period were unaffected by treatment. Prior to mating (Day 57) mean body weights for the control, low, intermediate and high dose females, respectively were 248.2, 244.6, 250.7 and 249.0 grams.

Mating and Fertility of F0 Animals:
The majority of control and treated animals copulated within the first few days, indicating that there were no problems with libido. There was no compound-related effect on gestation length as most dams delivered between day 21 and 22 of pregnancy.
Mean body weights for dams during gestation and lactation were unaffected by treatment. Mean body weight increases during gestation were 53.5%, 52.0, 49.3 and 49.8% for the control to high dose groups, respectively. Body weight gains during lactation were 9.1, 12.9, 9.3 and 8.3% for the control to high dose groups, respectively.
Pregnancy rates expressed as the number pregnant versus number cohabited were 86.4% (38/44), 84.4% (38/45), 88.9% (40/45) and 88.9% (40/45) for the control, low, intermediate and high dose groups, respectively. Collectively, of 179 animals mated, 23 were not pregnant, and since the distribution of these females was 6, 7, 5 and 5 in the control, low, intermediate and high dose groups, respectively, there was no indication of a drug effect. Lack of pregnancy was apparently not the result of decreased libido, since all non-pregnant animals had copulated as evidenced by the presence of either sperm or a deep seated plug in the vagina. Inspection of uteri at necropsy revealed that must of the 23 non-pregnant females had no implantation sites or other evidence of conception.
The value for mean number of implantation sites per dam (about 14) was about the same across all 4 treatment groups. However, mean numbers of pups born alive were slightly less for the high and intermediate dose groups compared to the low dose and control groups. This corresponded to postimplantation loss values of 10.3% and 10.6% for the high and intermediate doses, compared to 5.7% and 7.6% for the low and control groups, respectively. The number of litters in each dose group with less than 10 pups were 0, 1, 5 and 6 from control to high dose, respectively.

F1 Pup Survival and Body Weights:
Mean values for number of pups surviving during lactation and the corresponding individual data indicate that during the lactation period survival was not affected in any group. Specifically, the pup survival index on day 4 of lactation, which is the ratio of the mean number of pups on day 4, before culling, divided by the mean number of pups born alive, was similar for ail treatment groups. Similarly, the lactation index (mean number of pups alive on day 21 divided by the mean number of pups alive on day 4, post culling, showed no differences among the groups tested.
Treatment had no effect in pup weights as mean values on day 21 were 43.4, 39.5, 42.5 and 42.2 g respectively for the control, low, intermediate and high dose groups.
Gross external and internal examination of the F0 males and females revealed no treatment-related abnormalities. After selection of F1 pups needed for production of the F2 generation, the remaining F1 pups were sacrificed. External and internal examinations of these animals were unremarkable.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High dose level

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

General Observations Pertaining to F1 Animals:
High dose male #200, high dose female #218 and intermediate dose female #157 died during the treatment period prior to mating (about 4 weeks after weaning). Another high dose male (#198) was found dead during the treatment period prior to mating (about 10 weeks after weaning). Gross findings at necropsy of all 4 animals indicated dosing accidents as the cause of death. Generally, treatment was well tolerated as there were no obvious drug-related changes in appearance or behavior of the F1 animals during production of the F2a generation.

Body Weights and Food Consumption of F1 Animals Prior to the First Mating:
Body weight gains and food consumption of F1 animals were not affected by treatment, since within each sex values were similar between control and treated groups from the start of the growth period up to the point of mating.

Mating and Fertility of F1 Animals (First Mating):
The data of the number of days elapsed before observing evidence of mating do not offer an indication of a compound induced effect on libido. There was no drug-related effect on gestation length as most dams delivered between day 21 and 22 of pregnancy.
Body weight increases during gestation for the control to high dose groups respectively were 51.9%, 49.4%, 51.6% and 53.2%. During lactation, the high dose dams' average weight gain was 3.6%, while during the same period the control, low and intermediate dose dams had weight gains of 6.3%, 8.5% and 7.0%.
Pregnancy rates for the control, low, intermediate and high dose groups were 70% (21/30), 55.2% (16/29), 79.3% (23/29) and 72.4% (21/29), respectively. These values do not indicate any treatment effect on pregnancy rates.
At parturition the low dose group delivered about 20% fewer live pups (10.1/litter) than the control, intermediate or high dose groups whose litter sizes (12.2 to 13.4/litter) were similar to each other.

F2a Pup Survival and Body Weights:
The day 4 survival, and lactation indices were similar between all four groups.
Pup weights at birth were unaffected by treatment; 5.9, 5.8, 5.9 and 6.0 grams for the control to high dose groups, respectively. At 21 days of age the mean pup weights for the high dose group was 11% below control; 34.4 grams compared to 38.6, 38.4 and 37.8 grams for the control, low, and intermediate dose groups, respectively. In the high dose group, at day 21, there were 9 of 17 litters with 1 or more small pups (weight<30 grams) compared to 2 of 19 in the control group, 3 of 14 in the low dose and 5 of 20 in the intermediate dose.
Gross internal and external examinations of F2a pups revealed no apparent treatment-related abnormalities, except for the increased incidence of runts at the high dose.

Postnatal Development of F2a Pups:
One male and female pup per litter were tested for locomotor activity on day 21 of lactation. There were no statistically significant differences between the treated and control groups. All pups tested for auditory function responded with Preyer's reflex, and no ocular abnormalities were found during ophthalmoscopic examinations.

General Observations Pertaining to F1 Animals:
Generally, continued treatment of F1 rats during this part of the study did not affect general appearance or behavior.

Body weights and Food consumption of F1 animals prior to the second mating:
Body weight and food consumption were unaffected in the F1 animals prior to the second mating and only one death occurred during this period.

Mating and Fertility of F1 animals (second mating):
The data of pregnancy status and the number of days which elapsed before copulatory signs showed that treatment had no apparent effect on libido. Most of the dams that delivered pups did so between day 21 and 22 of pregnancy. However, about 50% of the dams in each treatment group did not deliver pups and were sacrificed on day 24-26 of gestation.
Percent increases in body weight during gestation for the control, low, intermediate and high dose, respectively, were 44.9%, 44.4%, 46.3% and 51.6%. Although there was no obvious treatment effect, the groups with values in the 45% range are slightly lower than values of 50%, observed previously. During lactation, the percent gains in mean body weight for the control, low, intermediate and high dose groups, respectively, were 5.7%, 6.7%, 5.0% and 3.1%. Similar values were observed for the F2a progeny, with the high dose females showing the smallest body weight increase.
Pregnancy rates for the control, low, intermediate and high dose groups (expressed as percent of cohabitated females) were 35.7% (10/28), 46.4% (13/28), 50.0% (13/26) and 57.1% (16/28), respectively. Despite the fact that most females showed signs of copulation, a relatively large percentage of animals did not conceive. Of 36 females that did not become pregnant during the first mating (F2a), 27 (75%) did not produce F2b offspring. Most of the females sacrificed after 24-26 days of gestation were barren with no evidence of resorptions or any uterine abnormalities. Of the 36 males which failed to impregnate females in the first (F2a) mating, 24 (67%) were again unsuccessful in the second mating (F2b).
Overall, the results indicate that the low pregnancy rates were more a result of failure to conceive rather than failure to copulate, and that the effect did not correlate with treatment. The decreased pregnancy rates are not totally unexpected. It is well known that in rodents fertility can diminish rapidly with increasing age. The females at the time of the second mating were 6 months of age compared to 3-4 months of age for the previous matings.
At parturition, the mean numbers of live pups per litter for the control to high dose groups, respectively were 13.7, 10.6, 13.7 and 13.6.

F2b Pup survival and Body weights:
There were no apparent treatment related effects on F2b pups survival. Day 4 survival indices and lactation indices did not show any treatment related trends. Mean pup weights per litter on day 1 for the control to high dose group, respectively, were 5.7, 5.6, 5.7 and 5.7 grams. At day 4, there was a dose related trend for slightly lower pup weights. By day 21, mean pup weights per litter were 35.5, 34.7, 33.4 and 29.5 grams, respectively for the control, low, intermediate and high dose groups. The 17% body weight differential between high dose and control is consistent with the 11% differential in day 21 pup weight for the high dose F2a group.
Gross internal and external examinations of the F2b pups revealed no apparent treatment related abnormalities. Gross internal and external examination of F1 animals was unremarkable.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Gross pathological findings:

no effects observed

F2a Pup Survival and Body Weights:
The day 4 survival, and lactation indices were similar between all four groups.
Pup weights at birth were unaffected by treatment; 5.9, 5.8, 5.9 and 6.0 grams for the control to high dose groups, respectively. At 21 days of age the mean pup weights for the high dose group was 11% below control; 34.4 grams compared to 38.6, 38.4 and 37.8 grams for the control, low, and intermediate dose groups, respectively. In the high dose group, at day 21, there were 9 of 17 litters with 1 or more small pups (weight<30 grams) compared to 2 of 19 in the control group, 3 of 14 in the low dose and 5 of 20 in the intermediate dose.
Gross internal and external examinations of F2a pups revealed no apparent treatment-related abnormalities, except for the increased incidence of runts at the high dose.

Postnatal Development of F2a Pups:
One male and female pup per litter were tested for locomotor activity on day 21 of lactation. There were no statistically significant differences between the treated and control groups. All pups tested for auditory function responded with Preyer's reflex, and no ocular abnormalities were found during ophthalmoscopic examinations.

F2b Pup survival and Body weights:
There were no apparent treatment related effects on F2b pups survival. Day 4 survival indices and lactation indices did not show any treatment related trends. Mean pup weights per litter on day 1 for the control to high dose group, respectively, were 5.7, 5.6, 5.7 and 5.7 grams. At day 4, there was a dose related trend for slightly lower pup weights. By day 21, mean pup weights per litter were 35.5, 34.7, 33.4 and 29.5 grams, respectively for the control, low, intermediate and high dose groups. The 17% body weight differential between high dose and control is consistent with the 11% differential in day 21 pup weight for the high dose F2a group.
Gross internal and external examinations of the F2b pups revealed no apparent treatment related abnormalities. Gross internal and external examination of F1 animals was unremarkable.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Dose descriptor:

NOEL

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

not specified

Conclusions:

Overall, compound administration did not alter the appearance or survival of the dams or sires. No treatment related differences were observed in pregnancy rates, litter size or pup survival. The only treatment related findings were 11% and 17% decrements in pup weight gain during lactation for the F2a and F2b offspring respectively, at the 1.0 mg/kg dose level.
Based on the finding in this 2-generation study with test substance, the No-Observed-Effect-Level (NOEL) is 0.3 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

1 (reliable without restriction)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Three fetotoxicity studies are available on rats, mice and rabbits.

Rat study (Kessedjian, 1989):

The test substance, an antiparasitic agent for treatment of both internal and external parasites in cattle, when administered orally to pregnant rats during organogenesis at daily dose levels of 1.5, 3 and 6 mg/kg, was not toxic to the dams. The compound was neither embryotoxic nor teratogenic at the three dose levels used.

Mouse study (Kessedjian, 1989):

The test substance, an novel avermectin antiparasitic agent when administered to pregnant mice in daily dose levels of 1.5, 3 and 6 mg/kg during organogenesis was not toxic to the dams. The high dose, however, was probably at the threshold for embryotoxicity. There were no evidence of terotogenicity. The no-effect level of toxicity in this study is 3 mg/kg/day.

Rabbit study (Kessedjian, 1988):

The test substance, when orally administered at 0.75 or 1.5 mg/kg to pregnant female rabbits during organogenesis was not considered to be toxic to dams or fetus, although some delay in ossification of fetal public bones occured at the latter dose. The dose level of 3 mg/kg induced maternal toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 31 May to 23 Jun 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP.

Qualifier:

according to guideline

Guideline:

other: Standard Procedures of the Centre de Recherche, Amboise

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charies River, St-Aubin-Les-Eibeuf, France
- Age at study initiation: 80 days (mean)
- Weight at study initiation: 226.5±10.6 (mean, grams±SD)
- Fasting period before study:
- Housing: individually on decontaminated (autoclaved) sawdust bedding (Litalabo, Societe Parisienne des Sciures, France) in makrolon cages (425x266x150 mm)
- Diet (e.g. ad libitum): Animals had free access to a pelleted commercial laboratory animal food (diet A04C, from Usine d'Alimentation Rationnelie, Villemolsson-sur-Orge, France)
- Water (e.g. ad libitum): Animals had free access to tap water.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3
- Humidity (%): 60±20
- Air changes (per hr): 14 times per hour
- Photoperiod (hrs dark / hrs light): From 7:00H to 19:00H.

IN-LIFE DATES: From 31 May, 1, 2, 3 Jun To 20-23 Jun 1988

Route of administration:

oral: gavage

Vehicle:

other: Sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0.15, 0.3, 0.6 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): A21606, A21395
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were collected from the top, middle and bottom of the suspensions used to treat the animals, before gavage. They were extracted and diluted by the mobile phase used for HPLC, and analysed in triplicate.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: one male for several females
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug referred to as day 0 post-insemination (p.i.)

Duration of treatment / exposure:

10 days (Day 6 to day 15 p.i.)

Frequency of treatment:

Once daily

Duration of test:

From day 0 to day 20 post-insemination (p.i.)

Remarks:

Doses / Concentrations:
1.5, 3 and 6 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in the present study were the same as those in maternal toxicity study (protocol 88034) where the dose of 6 mg/kg was considered as the no observable effect level (NOEL) in this species.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily during the treatment period and once daily at week-ends and before and after the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Main study: on days 1, 3, 6, 9, 12, 16, 18 and 20 p.i.
FOOD CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 20 p.i. in the morning
- Organs examined: Ovaries and uterine

OTHER:

Ovaries and uterine content:

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

Fetal examinations:

All fetuses were carefully examined for external and buccal malformations and weighed. Then the fetuses were numbered and sexed before killing by carbon dioxide. The skeletons of alternate fetuses from each litter were examined for the degree of ossification and for the presence of anomalies and the ribs were counted to detect presence of eventual supernumerary ribs (14th right, left or bilateral ribs).
The remaining fetuses were fixed in Bouin-Allen fluid before being serially hand-sectioned by the slicing technique of Wilson (1965). The slices were examined for visceral abnormalities or anatomical variations.

Statistics:

A covariance analysis was carried out on days 12, 16, 18 and 20 p.i. body weight of those pregnant females which produced at least one viable fetus, taking the day 6 p.i. body weight as covariates. Student's t-tests were used to compare the mean value of each treated group (adjusted for covariance) with that of the control group; the estimates of residual variance were used in the Student's t-test. The day 6 p.i. body weights were also analysed using anlysis of variance and comparing the treated group means with those of the controls by Student's t-test; the estimates of residual variance were used in the Student's t-test.
Analysis of variance (F test) was also used to analyse the number of corpora lutea, the number of implantation sites and the number of live fetuses.
Statistical analysis of fetal weights was carried out by the method described by Healy (1972), taking into account both the between and the within litter variability. The mean litter weights are calculated using individual fetal data. The adjusted means were weighted, taking into account the litter size and within litter variability (litters showing high variability have less impact on adjusted treatment means). Each of the treated groups was compared with controls using the approximate t-test on standard errors of adjusted means.

Indices:

None stated

Historical control data:

Yes

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no deaths and no drug-related clinical signs. Statistical analysis of body weights performed during and after the treatment period showned an increased body weight in all three treated groups, compared to controls. There were no differences between control and treated groups with regard to the reproductive parameters.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal weights were not affected by the maternal treatment. Some external anatomical variations and anomalies were observed in both control and treated groups. At visceral examination, a ventricular septal defect was observed at 3 mg/kg in one fetus presenting external anomalies. There were no visceral malformations in control, low and high dose groups. Skeletal examination revealed a slight delay of ossification of the 5th metacarpus and hyoid bone in the three treated groups. The percentage of fetuses with deformed ribs (wavy ribs) or rudimentary 13th rib(s) was increased in treated groups: wavy ribs 1.4%, 0% and 4.3% at 1.5, 3 and 6 mg/kg respectively versus 0.9% in controls; rudimentary ribs 5.1%, 7.7% and 4.3% at 1.5, 3 and 6 mg/kg respectively versus 1.8% in controls.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The test substance, an antiparasitic agent for treatment of both internal and external parasites in cattle, when administered orally to pregnant rats during organogenesis at daily dose levels of 1.5, 3 and 6 mg/kg, was not toxic to the dams.
The compound was neither embryotoxic nor teratogenic at the three dose levels used.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 16 Jun to 12 Jul 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP.

Qualifier:

according to guideline

Guideline:

other: Standard Procedures of the Centre de Recherche, Amboise

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

ICR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charies River, St-Aubin-Les-Eibeuf, France
- Age at study initiation: about 55 days
- Weight at study initiation: 27.35±1.7 (mean, grams±SD)
- Fasting period before study:
- Housing: individually on decontaminated sawdust bedding (Litalabo, Societe Parisienne des Sciures, France) in makrolon cages (267x207x140 mm)
- Diet (e.g. ad libitum): Animals had free access to a pelleted commercial laboratory animal food (diet A04C, from Usine d'Alimentation Rationnelie, Villemolsson-sur-Orge, France)
- Water (e.g. ad libitum): Animals had free access to tap water.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3
- Humidity (%): 60±20
- Air changes (per hr): 14 times per hour
- Photoperiod (hrs dark / hrs light): From 7:00H to 19:00H.

IN-LIFE DATES:

Route of administration:

oral: gavage

Vehicle:

other: Sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0.06, 0.12, 0.24 mg/mL
- Amount of vehicle (if gavage): 25 mL/kg bw/day
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the solutions used to treat the animals were analysed in triplicate by HPLC.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: one male for several females
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug referred to as day 0 post-insemination (p.i.)

Duration of treatment / exposure:

8 days (Day 6 to day 13 p.i.)

Frequency of treatment:

Once daily

Duration of test:

From day 0 to day 18 post-insemination (p.i.)

Remarks:

Doses / Concentrations:
1.5, 3 and 6 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in the present study were the same as in the prelininary fetotoxicity study (protocol 88035) in which a slight increase of embryomortality was observed at 6 mg/kg.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily during the treatment period and once daily at week-ends and before and after the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Main study: on days 1, 3, 6, 9, 12, 14 and 18 p.i.

FOOD CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 18 p.i. in the morning
- Organs examined: Ovaries and uterine

OTHER:

Ovaries and uterine content:

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

Fetal examinations:

All fetuses were carefully examined for external and buccal malformations and weighed. Then the fetuses were numbered and sexed before killing by carbon dioxide. The skeletons of alternate fetuses from each litter were examined for the degree of ossification and for the presence of anomalies and the ribs were counted and their degree of ossification were evaluated by the presence or absence of supernumerary ribs.
The remaining fetuses were fixed in Bouin-Allen fluid before being serially hand-sectioned by the slicing technique of Wilson (1965). The slices were examined for visceral abnormalities or anatomical variations.

Statistics:

A covariance analysis was carried out on days 12, 14 and 18 p.i. body weight of those pregnant females which produced at least one viable fetus, taking the day 6 p.i. body weight as covariates. Student's t-tests were used to compare the mean value of each treated group (adjusted for covariance) with that of the control group (adjusted for covariance); the estimates of residual variance were used in the Student's t-test. The day 6 p.i. body weights were also analysed using anlysis of variance and comparing the treated group means with those of the controls by Student's t-test; the estimates of residual variance were used in the Student's t-test.
Analysis of variance (F test) was also used to analyse the number of corpora lutea, the number of implantation sites and the number of live fetuses.
A chi-square test was used to analyse the embryomottality rate.
Statistical analysis of fetal weights was carried out by the method described by Healy (1972), taking into account both the between and the within litter variability. The mean litter weights are calculated using individual fetal data. The adjusted means were weighted, taking into account the litter size and within litter variability (litters showing high variability have less impact on adjusted treatment means). Each of the treated groups was compared with controls using the approximate t-test on standard errors of adjusted means.

Indices:

None stated

Historical control data:

Yes

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no deaths and no drug-related clinical signs. Statistical analysis of body weights performed during and after the treatment period showned an increased body weight in all three treated groups, compared to controls. There were no differences between control and treated groups with regard to the reproductive parameters.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal weights were not affected by the maternal treatment. Some external anatomical variations and anomalies were observed:
-fixed paws: control (2 fetuses), 1.5 mg/kg (1), 3 mg/kg (3)
-coelosomia: 1.5 mg/kg (1)
-cleft palate: 1.5 mg/kg (1), 3 mg/kg (1), 6 mg/kg (1)
-deformed digits: 3 mg/kg (1)
-syndactylia: 6 mg/kg (1)
-haematoma on the back: 6 mg/kg (1).
The number of fetuses with supemumerary ribs was comparable in control and treated groups. No visceral and no severe skeletal malformations were observed in fetuses of control or treated groups.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The test substance, an novel avermectin antiparasitic agent when administered to pregnant mice in daily dose levels of 1.5, 3 and 6 mg/kg during organogenesis was not toxic to the dams. The high dose, however, was probably at the threshold for embryotoxicity. There were no evidence of terotogenicity. The no-effect level of toxicity in this study is 3 mg/kg/day.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 04 Jul to 05 Aug 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP.

Qualifier:

according to guideline

Guideline:

other: Standard Procedures of the Centre de Recherche, Amboise

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Interfauna, U.K.
- Age at study initiation:
- Weight at study initiation: 3.61±0.17 (mean, kg±SD)
- Fasting period before study:
- Housing: separately in metal cages (size 50x56x38 cm), on wire grid floors, in a room separate from animals of other studies.
- Diet (e.g. ad libitum): Animals were given daily 150 grams of a pelleted commercial laboratory animal food (diet 112C, from Usine d'Alimentation Rationnelie, Villemolsson-sur-Orge, France)
- Water (e.g. ad libitum): Animals had free access to tap water.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18±3
- Humidity (%): 60±20
- Air changes (per hr): 14 times per hour
- Photoperiod (hrs dark / hrs light): From 7:00H to 19:00H.

IN-LIFE DATES: From 4 to 8 Jul To 1 to 5 Aug 1988

Route of administration:

oral: gavage

Vehicle:

other: Sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0.75, 1.5, 3 mg/mL
- Amount of vehicle (if gavage): 1 mL/kg bw/day
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were collected from the top, middle and bottom of the suspensions used to treat the animals, before gavage. They were extracted and diluted by the mobile phase used for HPLC, and analysed in triplicate.

Details on mating procedure:

Human chorionic gonadotrophin hormone (25 I.U., i.v.) was used one hour before aritificial insemination to induce ovulation respectively. The females were artifically inseminated with heterospermic samples of semen collected from male rabbits of the same strain and obtained from the same breeder. The day of insemination was called day 0 post insemination (p.i.). Induction of pseudo-gestation and insemination were staggered over a 5-day period at a rate of 16 females a day.

Duration of treatment / exposure:

12 days (Day 7 to day 18 p.i.)

Frequency of treatment:

Once daily in the morning

Duration of test:

From day 0 to day 28 post-insemination (p.i.)

Remarks:

Doses / Concentrations:
0.75, 1.5 and 3 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen according to the results of the maternal toxicity study in rabbits (protocal 88077) performed at 1.5, 3 and 6 mg/kg and in which a body weight decrease was observed in dams and fetuses at 6 mg/kg, when compared to controls.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily during the treatment period and once daily at week-ends and before and after the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Main study: on days 1, 3, 7, 9, 12, 15, 19, 21, 24 and 28 p.i.

FOOD CONSUMPTION: Twice daily during the treatment period and once daily at week-ends and before and after the treatment period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 28 p.i. in the morning
- Organs examined: Ovaries and uterine

OTHER:

Ovaries and uterine content:

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

Fetal examinations:

All fetuses were carefully examined for external and buccal malformations and weighed. Then the fetuses were numbered and killed by an intraperitoneal injection of sodium pentobarbitone (Dolethal). The skeletons of alternate fetuses from each litter were examined for the degree of ossification and for the presence of anomalies and the ribs were counted and their degree of ossification were evaluated by the presence or absence of supernumerary ribs.
The remaining fetuses were fixed in Bouin-Allen fluid before being serially hand-sectioned by the slicing technique of Wilson (1965). The slices were examined for visceral abnormalities or anatomical variations.

Statistics:

A covariance analysis was carried out on days 12, 19, 24 and 28 p.i. body weight of those pregnant females which produced at least one viable fetus, taking the day 7 p.i. body weight as covariates. Student's t-tests were used to compare the mean value of each treated group (adjusted for covariance) with that of the control group (adjusted for covariance); the estimates of residual variance were used in the Student's t-test. The day 7 p.i. body weights were also analysed using anlysis of variance and comparing the treated group means with those of the controls by Student's t-test; the estimates of residual variance were used in the Student's t-test.
Analysis of variance (F test) was also used to analyse the number of corpora lutea, the number of implantation sites and the number of live fetuses.
A chi-square test was used to analyse the embryomottality rate.
Statistical analysis of fetal weights was carried out by the method described by Healy (1972), taking into account both the between and the within litter variability. The mean litter weights are calculated using individual fetal data. The adjusted means were weighted, taking into account the litter size and within litter variability (litters showing high variability have less impact on adjusted treatment means). Each of the treated groups was compared with controls using the approximate t-test on standard errors of adjusted means.

Indices:

None stated

Historical control data:

Yes

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There were no deaths and no drug-related clinical signs. The matertnal body weights at 0.75 and 1.5 mg/kg were not statistically different from that of controls. At 3 mg/kg the maternal body weight was impaired during and after the treatment period (p<0.05 on day 7 p.i.; p<0.01 on day 12 p.i.; p<0.001 on days 19, 24 and 28 p.i.). There were no changes in the food consumption of the low dose group while at the mid and top dose levels, 4/18 and 14/19 females respectively showed a reduced food consumption on several occasions. The reproductive parameters were similar between control and treated groups.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no differences between control and treated groups with regard to the mean fetal weights. At external examination of fetuses, no anomalies were observed in control, low and mid dose groups. In the top dose group, 3 fetuses in the same litter (F811) had a cleft palate; in addition, one of these fetuses presented encephalocoelia. In another litter (F820), one fetus had phocomelia, syndactylia and coelosomia. There were no severe skeletal anomalies among the fetuses examined. The number of fetuses with delayed ossification of pubic bones was slightly increased at 1.5 and 3 mg/kg, compared to controls. Visceral examination revealed three cases of ventricular septal defect (one in each treated group) and one case of diaphragmatic hemia at the top dose in the fetus with coelosomia, phocomelia and syndactylia.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The test substance, when orally administered at 0.75 or 1.5 mg/kg to pregnant female rabbits during organogenesis was not considered to be toxic to dams or fetus, although some delay in ossification of fetal public bones occured at the latter dose. The dose level of 3 mg/kg induced maternal toxicity.
The dose of 0.75 mg/kg can be considered as the no-observable-effect-level (NOEL).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.75 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

1 (reliable without restriction)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on two-generation study and fetotoxicity studies results on animals, this substance has suspected reproductive toxicity to human and effect on or via lactation.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.7.1 (a) and 3.7.1 (b), this substance should be classified as Reproductive toxicant Category 2 and Effect on or via lactation.