N,N'-bis(2,2,6,6-tetramethyl-4-piperidyl)isophthalamide

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform GLP study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 206 - 211 g (males), 144 - 150 g (females)
- Housing: in transparent macrolon cages (type III), 1 animal per cage; one male / one female per cage (mating period); female with pups per cage (lactation period)
- Diet (e.g. ad libitum): sniff R/M-Z (V 1324), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 50+/-20%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Tylose H4000 G4 PHA (0,5% in deion. water)

Details on exposure:

VEHICLE
- Concentration in vehicle: 0, 12, 36 and 108 mL/kg
- Amount of vehicle (if gavage): 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. three weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 21 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): one per cage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from all suspensions prepared for treatment in weeks 6 and 12. Three samples (top, middle and bottom) were taken at each sampling date from each dose concentration analyzed (HPLC) for content within the stability period.

Duration of treatment / exposure:

16-18 weeks in total:
10 weeks during premating period (P-males and females)
1-3 weeks during mating (P-males and females)
3 weeks during gestation (P-males and females)
P-females: until end of lactation
P-males until scheduled sacrifice

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
60 mg/kg bw /d
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
180 mg/kg bw/d
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
540 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

28 males/females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
based on an acute oral toxicity test (LD50 1258 mg/kg bw) and a 28 day oral toxicity study (NOAEL 500 mg/kg bw/d)

Positive control:

none

Examinations

Parental animals: Observations and examinations:

- Mortality and clinical observations
All animals were examined before the start of the study and were shown to be in good general health condition. Mortality and morbidity were checked twice daily. Clinical observations were done at least once daily (at least least twice per day before parturition, length of gestation period was calculated), including once weekly detailed clinical examinations (neurological status, eyes mouth and teeth) in an open field.

- Body weight
Body weights were determined once weekly in both sexes during pre matin period and in females during gestation (weekly, days 0, 6, 13 and 20) and on day 0, 4, 7, 14 and 21 of the lactation period.

- Food consumption
Food consumption was recorded together with the body weights, i.e. at least once weekly.

Oestrous cyclicity (parental animals):

3 weeks before and during mating, daily vaginal smears were taken in order to determine estrus cycle length and normality. In addition, vaginal smears were taken once prior to scheduled necropsy.

Sperm parameters (parental animals):

no data

Litter observations:

Each litter born during the day was examined immediately after birth, those born in the night were examined on the following morning. Individual body weight, number and sex of still and live birth and the presence of gross anomalies of the offspring were noted. The day of birth was defined as day 0 of lactation.
The pups were observed for physical or behavioral abnormalities, the number, size and body weights of live pups recorded on days 0, 4, 7, 14 and 21.

Postmortem examinations (parental animals):

All P-generation and F1-offspring were necropsied and checked for macroscopically visible changes. Parental animals as well as all surviving pups culled on day 4 after birth, were killed on day 22 after birth. The remaining pups were killed on day 4 after birth. All abnormal findings were recorded.
The following tissues or organs of parental animals were preserved and processed for histopathological investigations:
epididymides, ovaries, pituitary, prostate, seminal vsicle, coagulating gland, testes, uterus, vagina and all fross lesions.
The above listed organs and tissues of the control and high dose parental (P) group animals at termination, from all scheduled deaths, as well as gross findings were embedded by conventional histological technique in Paraplast and stained with Hematoxylin-Eosin. In addition, F1-pups with abnormal findings were preserved in 10% formalin and grossly abnormal tissue from those processed for histopathologic examination.

Microscopic examination was performed on slides processed from vagina, uterus with cervix, ovaries, testes, epididymides, seminal vesicles, prostate, coagulating gland, pituitary gland from control and high dose parental animals, as well as on processed tissues with macroscopic observations. In the absence of treatment related changes, examination was not extended to the low and mid dose groups.

Postmortem examinations (offspring):

All P-generation and F1-offspring were necropsied and checked for macroscopically visible changes. Parental animals as well as all surviving pups culled on day 4 after birth, were killed on day 22 after birth. The remaining pups were killed on day 4 after birth. All abnormal findings were recorded. F1-pups with abnormal findings were preserved in 10% formalin and grossly abnormal tissue from those processed for histopathologic examination.

Statistics:

Inlife phase data of weekly body weight change values (from day 1 of dosing) were analysed for statistical significance within each sex by a 1-way ANOVA-based two-sided ordinal step-down trend test (Tukey et al., 1985). Inlife phase data of weekly food consumption values were analysed for statisitcal significance within each sex by a Jonckheere trend test with corrections for ties (Jonckheere, A.R.; 1954; Lin, F.O. and Haseman, J.K..; 1975) associated with a step-down procesure (Hothorn, L.; Lehmacher, W.; 1991).

Statisitcal evaluation on fertility data, pups and parental animals (post partum) was performed by Accovion, DM. R. Uhl:
A momotonic dose/repsonse relationship was assumed for the analysis of multigeneration reproductive toxicity studies. In the tested dose range, toxic effects observed at a specific dose level should also occur with any dose above that level. The below hypothesis tests were therefore embedded in a hierchical, closed testing "control treatment procedure" (Hothorn, L.; Lehmacher, W.; 1991).

Categorical data (i.e. the number of unreared litters per group) were analysed using one-sided Fisher's exact test (Fisher, R.A.; 1935). Ordinal data (e.g. counts or rates per animals) used one-sided Fisher's exact rank tests (Hollander, M.; Wolfe, D.A.; 1973).

Reproductive indices:

viabilty index (%), weaning index (%), survival rate (day 21)

Offspring viability indices:

no data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects 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 examined

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

- Dose formulation analysis
The achieved concentrations of the test substance in Tylose H 4000 G 4 PHA (0.5% in water) meet the acceptance criteria (85% - 115% for suspensions).

- Behavior, state of health and mortaliy (P-animals)
There were no unscheduled deaths observed, which could be related to administration of the test item in any treatment group. The animals were in a healthy condition throughout the entire study period.
There were three incidental and spontaeous deaths recorded: Female #93 (low dose group) was found dead on day 19. The cause of death could not be determined on macroscopic/microscopic level. Pregnant females #198 and #205 (high dose group) were found dead at the end of pregnancy (days 11 and 95, respectively), each with 20 normal developed fetuses. Female #198 had exhibited mammary adenoma which, however, was not the cause of death. The cause of death in those dams could not be determined, however, exhaustion due to the huge number of normal developed fetuses may be speculated to be the cause of death.

- Clinical oservations (P-animals)
Behavior and state of health remained unaffected by the administration of the test item in all dose groups throughout the observation periods (pre-mating, mating/pregnancy and lactation). There were not treatment related clinical signs. No opacity of the refracting media of the eyes, changes of the oral mucosa, or impairment of dental growth was observed.

- Body weight (P-animals)
Body weigths were not affected by the administration of the test item in all groups throughout the observation periods (pre-metaing, mating/pregancy, and lactation). body weights were slightly increased (ca. 10%) for females at 540 mg/kg bw/d during the pre-mating period, attaining statistical significance in weeks 1, 2, 3 and 6-10 as compared to the control. This finding was considered test item related, however, was considered not to be adverse. No corresponding finding was observed for those dams during pregnancy and the lacation phase.

- Food consumption (P-animals)
Food consumption remained unaffected by the administration of the test item throughout the study in all dose group males (pre-mating), mid and low dose group females (pre-mating) and all dose group females (pregnancy, lactation). Maen food consumption was occasionally slightly increased for females at 540 mg/kg bw/d, attaining statistical significance only in weeks 1, 2 and 6 in the pre-mating phase. Occasional statisitcal significant changes in relative food concsumption for males and females during pre-mating phase were considered not toxicologically significant.

- Estrus cylces and mating results
Individual estrus cycles measured during the last three weeks of the pre-mating period did not indicate any treatment related changes in estrus cycle length or normality in these groups. The mean number of estrus cycles within 3 weeks for the females was calculated to be 4.32, 3.81, 3.89, and 4.21 for the control, low-, mid- and high dose group, respectively.
There were no major differences in the mating attempts at 60 and 540 mg/kg bw/d as compared to the control. In these groups approx. 70% of the females became pregnant within the first week of the mating period. The slight difference in the mating attempt profile at 180 mg/kg bw/d (mid dose) was not dose related and hence considered to be incidental.
Accordingly, almost all females were recorded as being successfully mated within mean pre coital intervals of 4.8, 6.5, 8.16 and 6.5 days, respectively, which is within the physiological range of variation for this rat strain for groups 1, 2 and 4. For group 3, the maen pre coital time interval of 8.16 was slightly prolonged, however, in the absence of a dose response relationship, this finding was considered to be incidental.

- Macroscopic observations
animal #93 (group 2), #198 and #205 (both group 4) were found dead during the course of the experiment (day 19, day 111 or day 95, respectively). Female #198 had a mass on the mammary gland, however, the cause of death in any those animals could not be identified at macroscopic level.
Relevant gross pathology findings at scheduled necropsy were not observed, some spontaneous findings were as follows: a small testes and epididymis with deformation and changed consistency in one male of group 1 (control), a firm deposit in one epididymis of one male of group 2.

- Microscopic observations
There were no histopathological findings, which could be related to the administration of the test item in any of the animlas investigated. Incidental histopathological findings were as follows:
Atrophy of testis and epididymis and spermatocele was observed in one control male and spermatocele was een in another mid dose male.
A benign adenoma of the mammary gland was found in high dose female #198, which died during late pregnancy. this finding was incidental and correlated with the macro finding. It was not the cause of death in this animal.
Further sporadic findings were inflammatory reactions in the prostate of one animal in group 4, and a cyst in the pituitary gland in group 1.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

2

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

- Pregnancy and litter data
At birth, the number of pregnancies were 25, 23, 21 and 24 for control, low-, mid- and high dose females, respectively. Two high dose females spontaneously died during late pregnancy (day 20 and 22 p.c., respectively) and had to be excluded from statistical evaluation. There was one control female with dead pups at birth only. Hence, females at term with live pups counted 24, 23, 21 and 24, respectively, for low-, mid- and high dose group.
The mean number of implantation accounted 15.0, 15.8, 14.3 and 15.8, and hence, were comparable amongst all groups.
The mean live pups/litter accounted for 12.5, 13.7, 11.9 and 12.9, respectively, with birth indices of 85.9, 86.7, 81.0 and 82.4% for control, low-, mid- and high dose respectively, and hence, were comparable in all groups.
Body weight of high dose pups at birth was marginally but statisitcally significantly lower (<7%) as compared to the control. In the absence of any corresponding findings of body weight development during lactation, and the fact that this effect was confined to one sex, it was considered to be incidental.
The numbers of supernumery implantation sites were slightly higher in high dose dams as compared to the controls. However, the precentages of implantations were not statisitcally significantly different to the control, indicating that this finding was within the physiological range for this rat strain.
Mean gestation length was comparable in all groups, i.e. 23.4, 23.2, 23.3 and 23.4 for control, low-, mid- and high dose group, respectively.
Evaluation of the live offspring during lactation revealed no significant findings on viability index (%) on day 4, weaning index (%), and survival rate (day 21) in all treated groups including the high dose group when compared to the controls.
Accordingly, the mean body weight of live pups during lactation was comparable in all treated offspring to that of the control.
The number of dead pups counted 18, 18, 6 and 5, for control-up to the high dose group, and hance, was even lower in the mid and high dose groups as compared to the control and low dose group. Sex ratio (male vs. female) was 47.3, 51.7, 48.8 and 46.2% and hence within the physiological range for this rat strain and age.
No behavioral or other physical abnormalities were recorded for the offspring in any dose group including the high dose (540 mg/kg bw/d).

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

2

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

With regard to the present study the parental and offspring NOAEL and the NOEL are 540 mg/kg bw/d (highest dose tested). There was no evidence of reproductive toxicity in rats.

Executive summary:

The present study was conducted in order to determine the effects of N,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamide on reproductive performance when administered orally by gavage to male and female Sprague Dawley rats during pre mating, mating and lactation according to OECD 415 testing guidelines. The dose rationale was based on toxictiy data of a rat acute LD50 study and a subsequient rat 28 day oral (gavage) toxcity study.

Groups of 28 male and 28 female Sprague Dawley rats resceived the test item orally once daily at dose levels of 0, 60, 180 or 540 mg/kg bw/d for a period of 10 weeks prior to mating. Dosing of males was continued during the whole mating period until sacrifice (approx. week 16 -18 of the study). Dosing of females was continued during mating, and treatment for mated females continued during gestation, parturition, until day 21 of lactation. The dosing volume was 5 mL/kg bw corresponding to concentrations of 0, 12, 36 and 180 mg/mL.

Behavior and state of health were observed daily in all groups. Body weight development and food consumption were recorded throughout the study in females and during the pre mating period in males. 3 weeks before/during mating, daily vaginal smears were taken in order to determine estrus cycle length and normality, as well as additionally once prior to scheduled necropsy. During necropsy the animals were examined for macroscopically visible abnormalities with special emphasis on reproductive organs. Sexual organs, pituitary glands and all gross findings were preserved and processed for microscopic examination.

The dams were allowed to litter and rear their progeny to the stage of weaning. Growth, development and behavior of the offspring were assessed during lactation until scheduled necropsy. All pups were examined for external abnormalities.

Body weights, food consumption and litter parameters were analysed with the aid of a statistical program to show differences compared to the controls.

- Results in parental (P) animals:

There were no unscheduled deaths, which could be related to the administration of the test item. Three spontaneous deaths included one low dose female (day 19) and two high dose females at late pegnancy (days 95 and 111). The cause of death in these decedents could not be determined on macroscopic/microscopic level, and hence were considered to be incidental.

Apart form slightly higher body weight gains in high dose females (540 mg/kg bw/d) during the pre mating period, there were no test item related adverse findings in any group, including clinical signs, body weight and food consumption. There were no test item related findings recorded on estrus cycle, mating and reproductive performance and fertility.

Mean gestation length, rearing and development of their offspring remained unaffected by the administration of the test item. The mean gestation length was not altered by administration of the test item, Numbers of implantation, live or dead fetuses, supernumerary implantation sites and birth indices were not influenced by administration of the test item. The sex ratio of the pups was not altered by the administration of the test item.

There were no test item related findings at macroscopic or microscopic level.

- Results in the (F1) offspring:

Evaluation of the live offspring during lactation revealed no significant findings on viability indices (%), weaning index (%) and survival rate (day 21) in all treated groups compared to the controls.

Accordingly, the mean body weight of live pups during lactation was comparable in all treated offspring to that of the control. No behavioral or other physical abnormalities were recorded for the offspring in any dose group including the highest dose (540 mg/kg bw/d). There were no external abnormalities observed.

Daily oral (gavage) administration ofN,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamide to Sprague Dawley rats during the pre mating, mating, gestation and lactation period at dose levels of 60, 180 and 540 mg/kg bw/d did not affect general health, body weight development and food consumption. It did not affect male or female mating/reproductive performance, fertility, gestation length and development of their progeny, in the absence of parental toxicity.

With regard to the present study the parental and offspring NOAEL and the NOEL are 540 mg/kg bw/d (highest dose tested). There was no evidence of reproductive toxicity in rats.