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

Toxicity to reproduction

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Drug Safety Evaluation Standard Procedures

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(1'R,2S,4'S,5S,6R,8'R,10'E,12'S,13'S,14'E,16'E,20'R,21'R,24'S)-6-cyclohexyl-21',24'-dihydroxy-12'-{[(2R,4S,5S,6S)-5-{[(2S,4S,5S,6S)-5-hydroxy-4-methoxy-6-methyloxan-2-yl]oxy}-4-methoxy-6-methyloxan-2-yl]oxy}-5,11',13',22'-tetramethyl-5,6-dihydro-3',7',19'-trioxaspiro[pyran-2,6'-tetracyclo[15.6.1.1⁴,⁸.0²⁰,²⁴]pentacosane]-10',14',16',22'-tetraen-2'-one

EC Number:

601-490-4

Cas Number:

117704-25-3

Molecular formula:

C50H74O14

IUPAC Name:

(1'R,2S,4'S,5S,6R,8'R,10'E,12'S,13'S,14'E,16'E,20'R,21'R,24'S)-6-cyclohexyl-21',24'-dihydroxy-12'-{[(2R,4S,5S,6S)-5-{[(2S,4S,5S,6S)-5-hydroxy-4-methoxy-6-methyloxan-2-yl]oxy}-4-methoxy-6-methyloxan-2-yl]oxy}-5,11',13',22'-tetramethyl-5,6-dihydro-3',7',19'-trioxaspiro[pyran-2,6'-tetracyclo[15.6.1.1⁴,⁸.0²⁰,²⁴]pentacosane]-10',14',16',22'-tetraen-2'-one

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Batch No.: 15497-167-1
Purity: 92.5%

Test animals

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)

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

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

2

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

1

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

2

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

Results: F2 generation

General toxicity (F2)

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

Details on results (F2)

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.

Effect levels (F2)

1

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

2

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

Overall reproductive toxicity

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

not specified

Applicant's summary and conclusion

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.