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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

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 or developmental adeverse effects in rats.

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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

Remarks:

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

Remarks:

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

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

- 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.

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

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

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

- 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).

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

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

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

540 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable without restrictions.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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 of N,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.

Short description of key information:
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.
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.

Justification for selection of Effect on fertility via oral route:
This reproductive toxicity study performed with N,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamide was selected as the most relevant available reproductive toxicity study due to its reliability and since it provides a sensitive NOAEL.

Justification for selection of Effect on fertility via inhalation route:
In accordance with column 2 of REACH Annexes VIII and IX, the reproductive toxicity study does not need to use the inhalation route because exposure of human via inhalation, especially in a higher extent than via oral application as performed in the animal studies, is considered unlikely taking into account the vapour pressure/physical form of the substance (no inhalable particles) and intended use pattern (embedded in polymeric matrices). Oral uptake is the most likely exposure route

Justification for selection of Effect on fertility via dermal route:
In accordance with column 2 of REACH Annexes VIII and IX, the reproductive toxicity study does not need to use the dermal route because
- no primary systemic effects or other evidence of absorption were observed in skin and eye irritation studies in rabbits as well as in the sensitisation study in guinea pigs
- due to its physical form (handled as bloc) it is unlikely that higher amounts than tested in a repeated oral toxicity study will be systemically available via the intact skin barrier.

Effects on developmental toxicity

Description of key information

Developmental toxicity

1. OECD 414

An OECD 414 was performed with the test item. Nylostab S-EED was administered orally to pregnant razs from GD 5 to 19 at dose levels of 50, 250 and 1000 mg/kg bw/d.

There were no mortalities and clinical signs at all doses tested. There was a significant reduction in maternal body weights and food consumptions at 1000 mg/kg bw/d as compared to vehicle control group indicating maternal toxicity.The other maternal and litter data were comparable to vehicle control group up to the limit dose. Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all doses tested and revealed no signes of teratogenicity in any of the tested doses.

Based on these findings it is concluded that the NOAEL for maternal toxicity is 250 mg/kg bw/d, for fetal developmental toxicity 1000 mg/kg bw/d and for teratogenicity 1000 mg/kg bw/d.

A further 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.
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.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Clariant Plastics and Coatings(Deutschland) GmbH, BU Additives, Ludwig-Hermann-Strasse 100, 86368 Gersthofen, Germany

- Batch No.of test material: DEF2076375
- Expiration date of the batch: 17-May-2018
- Purity as per Certificate of Analysis: 98.65%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to + 25 °C)
- Stability under test conditions: The stability of the test item in the vehicle: 0.1%(w/v) Sodium Carboxymethyl cellulose (medium viscosity) in Milli-Q water was carried out under the Study No.: G12134. The test item was found to be stable and resuspendable in the vehicle for 4 days at room temperature, at the fortification levels of 1 mg/mL and 125 mg/mL.

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311,Gajwel Mandal, Medak District, Telangana

- Age at study initiation: 13 to 14 weeks
- Weight at study initiation:
Mean body weight(g) Body weight range(g)
G1 : 243.089 ± 18.37 210.12 to 268.16
G2 : 239.855 ± 19.42 204.58 to 268.84
G3 : 239.042 ± 20.79 206.35 to 267.97
G4 : 239.115 ± 21.14 208.16 to 268.57

- Housing: Rats were housed in standard polysulfone rat cages (size: Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube.
Following was the housing pattern during different periods of the experiment:
i. Pre mating / Acclimatization: Two rats of the same sex per cage were housed.
ii. Mating: Female rats were cohabited with males in a 1:1 ratio in same cage.
iii. Post-mating / Treatment: After mating confirmation, females were housed individually.
NOTE: The male rats and the remaining extra females were euthanized after mating procedure

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories, P.O.Box 44220, Madison Wi 53744-4220, was provided ad libitum to the rats.

- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: 08 December 2016 to 12 December 2016

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 64 – 66 %
- Air changes (per hr): 12 to 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 19 December 2016 To: 09 January 2017

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analyzed for active ingredient concentration (a.i.) and homogeneity at the initiation of treatment and at termination of treatment period.

The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another was kept as back up set which was stored in the experimental room depending upon the obtained stability results. For each set, two replicate samples were drawn from top, middle and bottom layers of each dose formulation. In case of control, two replicate samples from middle layer were drawn.
Samples were analyzed for Nylostab S-EED P content using analytical method which was validated under Advinus Study No. G12134. The analysis was carried out within the established stability period.

Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD, calculated using all the replicate values) of assay of top, middle and bottom layers (RSD) was equal to or less than 10.0 %.

The dose formulation suspensions were analyzed for active ingredient concentrations at the initiation and termination of treatment. The results of analysis of formulations revealed that the analyzed concentrations were within the acceptable limits

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 9 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy

Duration of treatment / exposure:

15 days

Frequency of treatment:

Gestation day 5 to Gestation day 19

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 2 (Low dose)

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 3 (Mid dose)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4 (High dose)

No. of animals per sex per dose:

Day '0" Pregnant rats : 24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: As per the Sponsor’s suggestion, based on the results of a 28 day repeated dose study, the following doses were selected for the main embryo-fetal developmental toxicity study with Nylostab S-EED P in Sprague Dawley rats by oral route:
• Vehicle control (G1): 0 mg/kg/day
• Low dose(G2) : 50 mg/kg/day
• Mid dose(G3) : 250 mg/kg/day
• High dose(G4) : 1000 mg/kg/day

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day (pre dose and post dose)
- Cage side observations checked in table [No.2] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: All females included in the study were weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20


OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [half per litter ]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female were analyzed using Analysis of Convariance (ANCOVA) taking litter size as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external, visceral and skeletal observations for variations were analyzed using Kruskal wallis test for group comparison.

The incidence of with and without resorptions in dams will be tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.

Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated as * throughout the report.

Indices:

MATERNAL DATA:
Maternal Parameters and Formula used

> Mean number of corpora lutea
Total no. of CL
= ----------------------------------------
Total no. of pregnant animals

> Mean number of implantations/group
Total no. of implantations
= ----------------------------------------
Total no. of pregnant animals

> Embryonic resorption index (%)
No. of early resorptions
= --------------------------------- x 100
No. of implantations

> Fetal resorption index (%)
No. of late resorptions
= -------------------------------- x 100
No. of implantations

> Pre-implantation loss per group (%)
No. of CL - No. of implantations
= ----------------------------------------------- x 100
No. of CL

> Post-implantation loss per group (%)
No. of (early + late) resorptions
= --------------------------------------------- x 100
Total no. of implantations

> Implantation index (%)
No. of implantations sites
= ------------------------------------- x 100
No. of corpora lutea

> Corrected body weight (Carcass weight)
= Terminal body weight (body weight on GD20) - unopened uterine weight.

> Corrected body weight gain
= Corrected body weight – body weight on GD5

LITTER DATA:
Litter Parameters and Formula used

> Mean litter size per group
Total No. of fetuses
= ---------------------------------
Total No. of pregnant animals

> Percentage of abnormal fetuses
No. of abnormal fetuses
= ---------------------------------- x 100
Total No. of fetuses

> Percentage of live fetuses (Live fetus index)
No. of live fetuses
= ----------------------------- x 100
Total No. of fetuses

> Percentage of dead fetuses (Dead fetus index)
No. of dead fetuses
= ----------------------------- x 100
Total No. of fetuses

> Sex ratio (F : M)
Number of females
= ---------------------------
Number of males

Historical control data:

The results of the study were discussed taking into consideration the historical data of this lab.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed throughout the experimental period 50, 250 and 1000 mg/kg/day dose groups.

Mortality:

no mortality observed

Description (incidence):

There was no morbidity or mortality throughout the experimental period in the 50, 250 and 1000 mg/kg/day dose groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The maternal group mean body weights were unaffected by the administration of test item Nylostab S-EED P at the doses of 50,
250 and 1000 mg/kg/day and were statistically comparable to vehicle control group.

At 50 and 250 mg/kg/day, the maternal body weight gain was comparable to vehicle control group during different periods of gestation.

At 1000 mg/kg/day, there was a significant reduction in maternal body weight gain during GD 14 to 17 (-21 %) and for entire gestation period (-14 %) as compared to vehicle control group.

The corrected body weight and corrected body weight gain was not statistically different from vehicle control group at all the tested doses.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The maternal food consumption was comparable to vehicle control group at all the doses tested during different periods of gestation, except for a significant reduction in food consumption during GD 5-8 (-15 %) at
1000 mg/kg/day.

The reduction in maternal body weights along with concomitant reduction in food consumption at 1000 mg/kg/day was considered as a treatment-related effect indicating maternal toxicity.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological findings in any of the tested doses

Number of abortions:

no effects observed

Description (incidence and severity):

The maternal data parameters comprising of gravid uterine weights, mean numbers of corpora lutea, implantations, early and late resorptions, pre and post-implantation loss rates and dams with any resorptions were unaffected by treatment with Nylostab S-EED P at all the doses tested.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

due to treatment-related significant reduction in maternal body weight gain and food consumption at 1000 mg/kg/day

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed during external observations of fetuses of dams treated up to 1000 mg/kg/day.

External malformations:

no effects observed

Description (incidence and severity):

Fetal, external observations were comparable to vehicle control group at all the doses tested.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Fetal, skeletal observations were comparable to vehicle control group at all the doses tested. Skeletal examinations revealed no signs of teratogenicity in any of the tested doses.

Visceral malformations:

no effects observed

Description (incidence and severity):

Fetal, visceral observations were comparable to vehicle control group at all the doses tested. Visceral examinations revealed no signs of teratogenicity in any of the tested doses.

Details on embryotoxic / teratogenic effects:

Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested doses.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Fetal developmental toxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

No Observed Adverse Effect Level (NOAEL) for:

- Maternal toxicity is 250 mg/kg/day due to treatment-related significant reduction in maternal body weight gain and food consumption at 1000 mg/kg/day, 

 - Fetal developmental toxicity is 1000 mg/kg/day as the litter data parameters were unaffected by treatment up to the high dose of 1000 mg/kg/day and

 - Teratogenicity is 1000 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of 1000 mg/kg/day

Table 1.       Details of the Experimental Design, Treatment Schedule, Maternal and Litter Data

Parameters	Group No.	G1	G2	G3	G4
	Dose (mg/kg/day)	0	50	250	1000
Total No.of rats found sperm positive/group		24	24	24	24
Duration of treatment(days)		GestationDays5to19(15days)
No.of rats sacrificed at caesarean section		24	24	24	24
No.of non-pregnantrats at caesarean section		1	3	0	0
No.of pregnant rats at caesarean section		23	21	24	24
No.of litters examined		23	21	24	24
Total no.of fetuses		289	266	288	270
Numberoffetusesevaluated
a. External examination		289	266	288	270
b. Visceral examination		144	133	144	135
c. Skeletal examination		145	133	144	135

 

Summary of Maternal Body Weight and Weight Gain

Table 2.              Summary of Maternal Group Mean Body Weight (g)

Group Mean body weight (g) on GD (gestation day)	Group No.	G1	G2	G3	G4
	Dose (mg/kg/day)	0	50	250	1000
	No. of rats	23	21	24	24
0	Mean	242.91	241.44	239.04	239.11
	SD	18.76	19.15	20.79	21.14
3	Mean	258.64	255.01	252.80	252.77
	SD	18.66	20.25	21.92	21.69
5	Mean	266.80	263.22	261.42	259.39
	SD	21.01	21.59	24.63	22.65
8	Mean	276.27	273.15	271.55	265.58
	SD	25.43	23.85	27.68	23.66
11	Mean	290.78	289.01	283.72	278.11
	SD	26.07	24.69	29.96	23.60
14	Mean	305.86	303.03	299.04	291.91
	SD	27.65	27.36	33.63	25.03
17	Mean	336.73	334.74	329.20	316.40
	SD	28.87	28.31	41.28	28.44
20	Mean	371.30	370.13	363.57	349.36
	SD	34.26	33.11	50.17	34.91

 

Table 3.              Summary of Maternal Body Weight Gain (g)

Period of treatment (days of gestation)	Group No.	G1	G2	G3	G4
	Dose (mg/kg/day)	0	50	250	1000
	No. of rats	23	21	24	24
0-3	Mean	15.73	13.57	13.76	13.66
	SD	4.99	4.30	4.15	4.53
3-5	Mean	8.15	8.21	8.62	6.62
	SD	5.06	3.97	5.42	2.92
5-8	Mean	9.47	9.93	10.12	6.19
	SD	6.24	5.14	5.46	4.44
8-11	Mean	14.51	15.86	12.17	12.54
	SD	4.37	3.54	5.21	4.39
11-14	Mean	15.08	14.02	15.32	13.79
	SD	4.41	5.08	5.87	4.70
14-17	Mean	30.87	31.71	30.17	24.50*
	SD	6.11	3.49	10.12	6.79
17-20	Mean	34.57	35.39	34.37	32.95
	SD	8.98	8.39	11.20	8.07

Values at each interval group Mean +/- SD; * significant different from G1

  

Table 3. contd.                Summary of Maternal Body Weight Gain (g)

Period of treatment (days of gestation	Group No.	G1	G2	G3	G4
	Dose (mg/kg/day)	0	50	250	1000
	No. of rats	23	21	24	24
Pre-treatment period (days 0-5)	Mean	23.88	21.78	22.38	20.28
	SD	8.00	6.76	6.76	6.0
Treatment period (days 5-20)	Mean	104.50	106.91	102.15	89.97
	SD	17.39	16.24	30.22	17.52
Entire gestation (days 0-20)	Mean	128.38	128.69	124.53	110.24*
	SD	21.53	18.33	33.71	18.27

Values at each interval group Mean +/- SD; * significant different from G1

 

Table 4.             Summary of Corrected Body Weight and Body Weight Gain (g)

Period of treatment (days of gestation	Group No.	G1	G2	G3	G4
	Dose (mg/kg/day)	0	50	250	1000
	No. of rats	23	21	24	24
Body weight on GD 5	Mean	266.80	263.22	261.42	259.39
	SD	21.01	21.59	24.63	22.65
Terminal Body weight on GD 20	Mean	371.30	370.13	363.57	349.36
	SD	34.26	33.11	50.17	34.91
Uterine weight	Mean	76.72	75.43	72.77	69.71
	SD	17.59	14.92	26.33	18.13
Carcass weight (corrected body weight on GD 20)	Mean	294.58	294.70	290.80	279.64
	SD	32.86	25.79	32.29	27.55
Corrected body weight gain	Mean	27.78	31.48	29.38	20.25
	SD	14.60	15.16	12.71	11.20

GD: Gestation Day

Corrected body weight on gestation day 20 (Caracass weight) = Terminal Body weight on GD 20 – unopended uterine weight.

Corrected body weight gain = caracass weight – body weight on Gestation day 5

Table 5.             Summary of Maternal data

Parameters	GroupNo. Dose(mg/kg/day)	G1 0	G2 50	G3 250	G4 1000
	No.of Rats	23	21	24	24
Gravid uterine weight(g)	Mean	76.72	75.43	72.77	69.71
	SD	17.59	14.92	26.33	18.13
NumberofCorpora lutea	Mean	15.65	15.76	15.96	14.88
	SD	2.40	3.08	4.42	2.98
Numberof Implantations	Mean	13.48	13.19	12.75	11.83
	SD	2.79	2.46	4.42	3.38
Early Resorptions	Mean	0.87	0.43	0.71	0.50
	SD	1.32	0.60	1.12	0.83
Late Resorptions	Mean	0.04	0.10	0.04	0.08
	SD	0.21	0.30	0.20	0.28
Pre-implantation Loss	Mean	2.17	2.57	3.21	3.04
	SD	2.39	2.87	3.18	3.43
Post-implantation Loss	Mean	0.91	0.52	0.75	0.58
	SD	1.35	0.60	1.29	0.83
DamswithanyResorption	Total	12	10	11	10

 

 

Table 5. contd. Summary of Maternal data (% per litter)

Parameters	Group No. Dose(mg/kg/day)	G1 0	G2 50	G3 250	G4 1000
	No.of Rats	23	21	24	24
Early Resorptions	Mean	7.18	3.54	8.11	3.98
	SD	13.93	4.97	17.03	6.33
Late Resorptions	Mean	0.26	0.87	0.60	0.97
	SD	1.23	2.77	2.92	3.39
Pre-implantation Loss	Mean	13.54	14.94	21.24	19.65
	SD	15.78	15.08	23.41	22.69
Post-implantation Loss	Mean	7.43	4.41	8.71	4.95
	SD	13.96	5.09	19.43	6.59
Implantation Index	Mean	86.46	85.06	78.76	80.35
	SD	15.78	15.08	23.41	22.69

 

Table 6.             Summary of Litter data

Parameters	Group No. Dose(mg/kg/day)	G1 0	G2 50	G3 250	G4 1000
	No.of Rats	23	21	24	24
No.of litters		23	21	24	24
Total no.of fetuses		289	266	288	270
Mean litter size		12.6	12.7	12.0	11.3
Total live fetuses
a.  Number		289	266	288	270
b.  Weight(g)	Mean	4.02	4.02	4.03	4.10
	SD	0.18	0.35	0.29	0.42
Live male fetuses
a.  Number		141	145	132	154
b.  Weight(g)	Mean	4.11	4.13	4.15	4.15
	SD	0.19	0.40	0.30	0.41
Live female fetuses
a. Number		148	121	156	116
b.  Weight(g)	Mean	3.93	3.89	3.87	3.94
	SD	0.20	0.35	0.24	0.42
Sex Ratio-Male:Female		1:1.05	1:0.83	1:1.18	1:0.75

 

Table 7.             Summary of Gross Pathological findings

Group No.	G1	G2	G3	G4
Parameters / Dose(mg/kg/day)	0	50	250	1000
1.     No. of rats subjected to caesarian section	24	24	24	24
2.     No.of rats pregnant at caesarian section	23	21	24	24
3.     No.of rats showing gross pathology	0	0	0	0

Table 8.              Summary of Fetal External Observations

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			50			250			1000
No. of litters examined	23			21			24			24
No. of fetuses examined	289			266			288			270
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Normal Variant	None
Minor Anomalies
Small fetus	0	0.00	0.00	1	0.38	1.30	0	0.00	0.00	0	0.00	0.00
Major Malformations	None

Table 9.              Summary of Fetal Visceral Observations (incidence and percentage)

 

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			50			250			1000
No. of litters examined	23			21			24			24
No. of fetuses examined	144			133			144			135
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Normal Variant
Kidney renal pelvis dila. (Rt/Lt/B) (+)	0	0.00	0.00	2	1.50	3.63	0	0.00	0.00	3	2.22	8.81
Minor Anomalies
Kidney renal pelvis dila. (Rt/Lt/B) (++)	3	2.08	6.55	0	0.00	0.00	3	2.08	7.14	0	0.00	0.00
Major Malformations	None

+: Slight

++: moderate

Table 10.           Summary of Fetal Skeletal Observations (incidence and percentage)

              

Group No.	G1			G2				G3			G4
Dose (mg/kg/day)	0			50				250			1000
No. of litters examined	23			21				24			24
No. of fetuses examined	145			133				144			135
	Inc.	Fetus %	Litter %	Inc.		Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.		Fetus %	Litter %
Normal Variant
DSO:  Stern: # 5	3	2.07	2.11	1		0.75	0.68	1	0.69	0.69	2		1.48	1.22
Stern: # 6	0	0.00	0.00	1		0.75	0.60	0	0.00	0.00	7		5.19	4.88
Stern: # 5,6	1	0.69	0.62	2		1.50	1.90	0	0.00	0.00	1		0.74	0.60
CV:centra: 1/7	0	0.00	0.00	0		0.00	0.00	1	0.69	0.60	0		0.00	0.00
CV:centra: 2/7	2	1.38	1.35	2		1.50	1.98	0	0.00	0.00	1		0.74	1.04
CV:centra: 3/7	4	2.76	2.69	1		0.75	1.19	2	1.39	1.19	0		0.00	0.00
CV:centra: 4/7	6	4.14	4.18	1		0.75	0.68	4	2.78	2.62	4		2.96	3.72
CV:centra: 5/7	31	21.38	20.05	19		14.29	14.82	22	15.28	14.93	18		13.33	13.52
CV:centra: 6/7	64	44.14	46.05	47		35.34	34.99	64	44.44	43.32	52		38.52	36.73
CV:centra: 7/7	38	26.21	25.68	63		47.37	46.34	51	35.42	33.17	60		44.44	40.82
DSO:  TV:centra:1/13	1	0.69	0.72	0	0.00		0.00	0	0.00	0.00		0	0.00	0.00
CdV:centra:1/4	3	2.07	1.79	14	10.53		9.15	7	4.86	3.41		21	15.56	12.67
CdV:centra:2/4	1	0.69	0.62	0	0.00		0.00	0	0.00	0.00		1	0.74	0.60
CdV:centra:4/4	0	0.00	0.00	1	0.75		0.95	0	0.00	0.00		0	0.00	0.00
CdV:arch:1/2	4	2.76	2.33	6	4.51		3.87	10	6.94	5.19		17	12.59	10.24
CdV:arch:2/2	0	0.00	0.00	1	0.75		0.95	0	0.00	0.00		0	0.00	0.00
F limb:Metacarp.:1/4	6	4.14	3.57	11	8.27		7.53	6	4.17	6.71		9	6.67	5.41
F limb:Pr. Phal:1/2	0	0.00	0.00	3	2.26		2.07	10	6.94*	5.82		11	8.15*	6.56
F limb:Pr. Phal:2/2	2	1.38	1.16	4	3.01		3.11	2	1.39	1.39		9	6.67	5.70
F limb:Dt. Phal:1/4	1	0.69	0.62	1	0.75		0.68	0	0.00	0.00		2	1.48	1.12

*: Significantly different from G1

Table 10. contd.              Summary of Fetal Skeletal Observations (incidence and percentage)

 

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			50			250			1000
No. of litters examined	23			21			24			24
No. of fetuses examined	145			133			144			135
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.		Fetus %	Litter %
Normal Variant
DSO: F limb:Dt. Phal:4/4	0	0.00	0.00	1	0.75	0.95	0	0.00	0.00	0		0.00	0.00
H limb:Dt. Phal:1/5	1	0.69	0.54	0	0.00	0.00	2	1.39	1.12	3		2.22	1.79
H limb:Dt. Phal:5/5	1	0.69	0.62	2	1.50	1.63		0.00	0.00	4		2.96	2.31
INO/PO:Stern:# 2,5	3	2.07	1.71	11	8.27	7.24	13	9.03	7.86	10		7.41	6.17
Stern:# 1	0	0.00	0.00	0	0.00	0.00	1	0.69	0.60	0		0.00	0.00
Stern:# 2	2	1.38	2.90	7	5.26	4.90	4	2.78	2.48	8		5.93	5.74
Stern:# 4	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00	1		0.74	0.52
Stern:# 5	24	16.55	14.61	18	13.53	13.02	27	18.75	16.40	30		22.22	19.11
Stern:# 6	14	9.66	8.35	23	17.29	15.70	15	10.42	8.22	13		9.63	8.13
CV:centra:1/7	24	16.55	15.10	12	9.02	8.76	19	13.19	12.22	18		13.33	12.54
INO/PO:CV:centra:2/7	1	0.69	0.62	2	1.50	2.38	2	1.39	1.43		2	1.48	1.64
CV:centra:3/7	1	0.69	0.72	1	0.75	0.79	0	0.00	0.00		0	0.00	0.00
TV:centra:1/13	0	0.00	0.00	1	0.75	0.68	1	0.69	0.42		0	0.00	0.00
Minor Anomalies
HYPOPLASTIC:    Stern:# 2	0	0.00	0.00	1	0.75	0.79	1	0.69	0.60		0	0.00	0.00
Stern:# 5	8	5.52	4.92	4	3.01	2.68	3	2.08	1.71		4	2.96	2.54
DB:  TV:centra:1/13	34	23.45	28.30	33	24.81	23.43	36	25.00	25.92		29	21.48	19.70
TV:centra:2/13	17	11.72	10.94	11	8.27	7.97	19	13.19	15.47		14	10.37	9.49
TV:centra:3/13	6	4.14	3.65	3	2.26	2.31	5	3.47	2.64		4	2.96	2.86
TV:centra:4/13	1	0.69	0.62	2	1.50	1.36	3	2.08	1.63		4	2.96	2.71

 Table 10. contd.              Summary of Fetal Skeletal Observations (incidence and percentage)

 

Group No.		G1			G2			G3			G4
Dose (mg/kg/day)		0			50			250			1000
No. of litters examined		23			21			24			24
No. of fetuses examined		145			133			144			135
		Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Minor Anomalies
DB: TV:centra:5/13		0	0.00	0.00	0	0.00	0.00	0	0.00	0.00	1	0.74	0.52
LV:centra:1/6		7	4.83	4.48	8	6.02	5.83	3	2.08	1.53	6	4.44	4.24
LV:centra:2/6		1	0.69	0.62	0	0.00	0.00	0	0.00	0.00	1	0.74	0.83
LV:centra:4/6		0	0.00	0.00	0	0.00	0.00	0	0.00	0.00	1	0.74	0.52
SPLIT:TV:centra:1/13		2	1.38	1.24	4	3.01	2.95	1	0.69	0.60	1	0.74	1.04
ASY DB:     TV:centra:1/13		0	0.00	0.00	1	0.75	0.79	0	0.00	0.00	4	2.96	2.82
TV:centra:2/13		0	0.00	0.00	1	0.75	0.68	0	0.00	0.00	0	0.00	0.00
ASY OSSI:   stern:#4,5		1	0.69	0.62	0	0.00	0.00	0	0.00	0.00	1	0.74	0.69
stern:# 3,4		0	0.00	0.00	0	0.00	0.00	1	0.69	0.60	1	0.74	0.52
stern:# 3-5		1	0.69	2.17	2	1.50	1.59	1	0.69	0.83	1	0.74	0.69
ASY SPLIT:TV:centra:1/13	1		0.69	0.62	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00
EXTRA: LV:centra & arch #7	0		0.00	0.00	0	0.00	0.00	0	0.00	0.00	1	0.74	0.69
RUDIMENTARY: RIB(Rt/Lt/B):# 14	46		31.72	31.60	43	32.33	32.30	21	14.58*	12.99	50	37.04	37.05
ACCESSORY: RIB(Rt/Lt/B):# 14	2		1.38	1.45	1	0.75	0.60	1	0.69	0.60	1	0.74	0.60
WAVY:RIB(Rt/Lt/B)(+/++):2/13	1		0.69	0.62	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00
Major Malformations	None

*: Significantly different from G1

+: Slight

++: moderate

Conclusions:

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

- Maternal toxicity is 250 mg/kg/day due to treatment-related significant reduction in maternal body weight gain and food consumption at 1000 mg/kg/day,

- Fetal developmental toxicity is 1000 mg/kg/day as the litter data parameters were unaffected by treatment up to the high dose of 1000 mg/kg/day and
- Teratogenicity is 1000 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of 1000 mg/kg/day

in Sprague Dawley rats when Nylostab S-EED P was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.

Executive summary:

The objective of this study was to evaluate theembryo-fetal developmentaltoxicity of test itemNylostabS-EED Pwhen administered to pregnant Sprague Dawley rats by oral route during gestation days (GD) 5 to GD19.The results of this study helped to establish the No Observed Adverse Effect Level (NOAEL) of the test item.

 

In this study, each group (G1, G2, G3 and G4) consisted of 24 presumed pregnant Sprague Dawley rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal plug was observed or vaginal smear was found sperm positive.

 

The test item,NylostabS-EED Pwas suspended in vehicle [0.1% (w/v) Sodium Carboxymethyl cellulose (medumviscosity) in Milli-Q water] and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 50, 250 and 1000 mg/kg/day for low (G2), mid (G3) and high (G4) dose group rats, respectively. The rats in the vehicle control (G1) group received the vehicle alone. A constant dose volume of 10 mL/kg body weight was administered to all groups.

 

The dose formulation suspensions were analyzed for active ingredient concentrations at the initiation and termination of treatment. The results of analysis of formulations revealed that the analyzed concentrations were within the acceptable limits.

 

The mated females were observed twice daily for clinical signs, mortality and morbidity. Body weights were recorded on GD0, 3, 5, 8, 11, 14, 17 and 20. About 200 g (food input) was provided on Day ‘0’. The food left over was recorded and replenished to about 200 g on GD 3, 5, 8, 11, 14 and 17. The food left over was also recorded on Day 20 of presumed gestation. The intermittent body weight gain and food intake was calculated and presented for rats found pregnant at caesarean section.

 

Caesarean section was performed for all the rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the dams was removed (by laparotomy) and the contents were examined.The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and deadfetuses. The number of corpora lutea was counted on each ovary. All thefetuseswere sexed, weighed and examined for external malformations.Approximately half the number of fetuses from each dam was examined for visceral malformations and the remaining half was evaluated for skeletal malformations.

 

Results of the study are presented below:

·        There were no mortalities and clinical signs at all the doses tested.

·        There was significant reduction in maternal body weights and food consumption at 1000 mg/kg/day as compared to vehicle control group indicating maternal toxicity.

·        The other maternal and litter data parameters were comparable to vehicle control group up to the high dose of 1000 mg/kg/day.

·        Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested doses.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

·           Maternal toxicity is 250 mg/kg/day due to treatment-related significant reduction in maternal body weight gain and food consumption at 1000 mg/kg/day, 

·           Fetaldevelopmental toxicity is 1000 mg/kg/day as the litter data parameters were unaffected by treatment up to the high dose of
1000 mg/kg/day and

·           Teratogenicity is 1000 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of 1000 mg/kg/day

 

in Sprague Dawley rats whenNylostabS-EED Pwas administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

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

Additional information

The present one generation reproductive toxcity 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 of N,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 or developmental toxicity in rats

Toxicity to reproduction: other studies

Additional information

no further data mandatory

Justification for classification or non-classification

There is no evidence to suggest that a classification for reproductive or developmental toxicity is appropriate.

With reference to the valid reproductive toxicity study according OECD 415 with the fully viable F1-generation resulting in the test design of a 1-generation study and the lack of reproductive/developmental effects and the outcome of the fully relaible OECD 414 study, it is concluded that N,N’-Bis(2,2,6,6-tetramethyl-4-piperidinyl)isophthalamidei s not subject to classification and labelling according to Regulation 1272/2008/EC regarding reproductive and developmental toxicity.