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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Reproduction/developmental toxicity screening test of NECTARYL in rats by dietary administration was performed in 2015 according to the OECD Guideline No. 421.

No reproductive or developmental effects were observed up to the highest dose tested. PArental toxicity was observed at the highest dose tested.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 February 2015 to 30 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
There were no deviations from standard operating procedures that affected the integrity of the study.
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Name as stated in the report: NECTARYL
Batch No.: VE00364047
Appearance: Colourless liquid
Expiration date: 09 January 2016
Species:
rat
Strain:
other: Rat: Crl:WI(Han) (outbred, SPF-Quality).
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. WIL Research Europe B.V. has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test system FO: Nulliparous and non-pregnant females and untreated rats (Crl:WI(Han)) were used at initiation of the study.
Source F0: Charles River Deutschland, Sulzfeld, Germany.
Age at start F0-treatment: Approximately 10-12 weeks.
Number of F0-animals: 40 females and 40 males.
Acclimatization F0: At least 5 days prior to start of treatment
Health inspection F0: Upon receipt of the animals.
Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0: Earmark and tattoo
Environmental conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap-water.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test substance was mixed directly with some powder feed without the use of a vehicle (premix) and subsequently mixed with the bulk of the diet. No correction was made for the purity or the specific gravity/density of the test substance.
Elix water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35ºC before storage. The control animals were received similarly prepared pellets but without the test substance.
The amount of test substance incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period.
The actual test substance intake was estimated based on the body weight and food consumption values.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Detection of mating was not confirmed for animal no. 52 (Group 2) which had implantation sites only. The mating date of this animal could not be determined.
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (11 February 2015), according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
In addition, random samples were taken from all diet preparations and stored at ≤-15ºC for possible future analysis. Any remaining samples were discarded after approval by the sponsor or at finalization of the study report. Sampling was performed as soon as possible after drying the pellets. After preparation, the samples were stored at ≤-15ºC until analysis.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed for 40-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
Ad libitum in diet
Details on study schedule:
Experimental starting date: 02 February 2015 (allocation)
Start treatment: 03 February 2015
Start mating: 17 February 2015
Delivery of litters: 12-16 and 24-25 March 2015
Necropsy: 04 March 2015 (males) and 15-20 and 30 March 2015 (females and pups)
Experimental completion date: 30 March 2015 (end of in-life phase)
Dose / conc.:
0 ppm (nominal)
Remarks:
Group 1 (10 males and 10 females)
Dose / conc.:
1 000 ppm (nominal)
Remarks:
Group 2 (10 males and 10 females)
Dose / conc.:
3 000 ppm (nominal)
Remarks:
Group 3 (10 males and 10 females)
Dose / conc.:
10 000 ppm (nominal)
Remarks:
Group 4 (10 males and 10 females)
No. of animals per sex per dose:
10 males and 10 females per group / 1 dose concentration per group
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on the results of the 28-Day study (Project 505866) where dose levels of 1000, 3000 and 10000 ppm were tested. An adverse effect on the liver was the primary finding. At 10000 ppm higher relative liver weights were seen (approximately 45% and 33% for males and females, respectively) at the end of treatment and also remained higher for males at the end of recovery. There were also changes in clinical biochemistry parameters that may be related to altered liver function including lower total protein level in females and higher cholesterol level in both sexes at 10000 ppm. At 3000 ppm, relative liver weights were approximately 17% higher for males. Other treatment related effects were seen, but none were considered adverse and thus were not mentioned further here. The NOAEL was set at 3000 ppm for both sexes.
Parental animals: Observations and examinations:
- Mortality / Viability: At least twice daily.
- Clinical signs: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
- Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
- Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.
- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
- General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Postmortem examinations (parental animals):
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands) :
Cervix, Clitoral gland, Preputial gland, Coagulation gland, Prostate gland, Epididymides , Seminal vesicles, Female mammary gland area, Testes , Kidneys, Thyroids, Liver, Uterus, Ovaries, Vagina
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
The only clinical sign noted was alopecia which was seen for a single female at 10000 ppm. This is incidental in nature and not attributable to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test substance.
One control female (no. 41) was found dead on Day 41 of treatment (Day 1 of lactation). No cause of death could be determined. As this was a control female, this was not attributable to treatment with the test substance.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were significantly lower at 10000 ppm than controls for both sexes during the premating and mating periods. Absolute body weights were also slightly lower than controls during this time, though the difference from controls was not statistically significant.
Females at 10000 ppm had significantly lower body weights and weight gains from Days 4 and 7 of the post coitum period, respectively. The significantly lower absolute weights persisted throughout the duration of treatment while weight gains normalized to control levels during lactation.
At 3000 ppm, absolute body weights were significantly lower for females on Day 1 of lactation only. As this occurred on a single time point, this was not considered to be toxicologically relevant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no adverse effects on food consumption with treatment up to 10000 ppm.

At 10000 ppm, relative food consumption was higher for males from Day 8 of the premating period throughout the remainder of treatment. Females at 10000 ppm had lower absolute and relative food consumption during the first week of treatment, but surpassed control levels from Days 8-15 of the premating period. Relative food consumption was higher for all treated females during the post coitum and lactation periods.

The lower food consumption initially seen for high dose females may reflect palatability issues with the diet. The increased food consumption for treated females may indicate that animals ate a greater volume of food to meet their nutritional needs as a larger proportion of the diet was composed of (nonnutritive) test item in the treated groups. The differences from controls were considered to be treatment related but not adverse.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were evident at 10000 ppm and included:
Thyroid gland: follicular cell hypertrophy was noted at an increased severity in 8/10 males of the 10000 ppm group (2 minimal, 6 slight) compared to 4/10 males of the control group (3 minimal, 1 slight), 3/10 of the 1000 ppm group (3 minimal) and in 6/10 of the 3000 ppm group (5 minimal, 1 slight).
Liver: hepatocellular hypertrophy was noted at an increased incidence and/or severity in 8/10 males (6 minimal, 2 slight) and 6/10 females (5 minimal, 1 slight) of the 10000 ppm group, compared to 2/10 females (2 minimal) of the control group, in 1/10 females (1 minimal) of the 1000 ppm group and in 2/10 females (2 minimal) of the 3000 ppm group.
Kidneys: a dose-dependent increase in incidence and/or severity of hyaline droplet accumulation was noted in 5/10 males of the 1000 ppm group (4 minimal, 1 slight), in 7/10 of the 3000 ppm group (5 minimal, 2 slight) and in 10/10 of the 10000 ppm group (7 minimal, 3 slight), compared to 0/10 in the control males.
There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
There were no test item-related microscopic finding, including spermatogenesis assessment, in the reproductive organs from the animals that did not produce offspring or did not mate at 1000 and 3000 ppm.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No toxicologically relevant effects on reproductive parameters were noted.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
Female no. 76 (10000 ppm), had slightly more pups than the number of implantation sites recorded. This was considered caused by normal resorption of these areas as the enumerations were performed on Day 5 of lactation.
Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The pups from litter no. 41 that were euthanized had less milk in the stomach and were cold to the touch. These were the only clinical signs seen for any pup apart from pup no. 1, litter no. 51 who was noted as pale and had a lean appearance at the first litter check.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were 14, 1 and 3 pups that died or went missing in the first days of lactation in the control, 1000 and 3000 ppm groups, respectively. Missing pups were most likely cannibalised. There were no pups that died or went missing at 10000 ppm. Twelve of the 14 pups in the control group were from the female that was found dead (no. 41). Importantly, of this litter, 11 of the 12 were living (one pup was dead at first litter check) but had to be euthanized after their mother was found dead.
No toxicological relevance was attributed to the dead/missing pups at 1000 and 3000 ppm since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were unaffected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No milk in the stomach was the only macroscopic finding noted for pups that were found dead. This was incidental. There were no macroscopic findings noted for pups surviving to the scheduled necropsy.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 10 000 ppm
Based on:
test mat.
Remarks:
corresponds to 743-750 mg/kg for males and 720-1226 mg/kg for females
Sex:
male/female
Basis for effect level:
other: no test item adverse effect observed in pups
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
720 mg/kg bw/day
Treatment related:
no
Conclusions:
In conclusion, treatment with NECTARYL by diet in male and female Wistar Han rats at dose levels of 1000, 3000 and 10000 ppm revealed parental toxicity at 10000 ppm. No reproductive or developmental toxicity was observed with treatment up to 10000 ppm.
Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 3000 ppm was derived and a reproductive and developmental NOAEL of at least 10000 ppm was determined.
When corrected for mean test article intake, the parental NOAEL of 3000 ppm corresponds to 208-214 mg/kg for males and 235-348 mg/kg for females. The reproductive and developmental NOAEL of 10000 ppm corresponds to 743-750 mg/kg for males and 720-1226 mg/kg for females.
Executive summary:

NECTARYL was administered by diet to male and female Wistar Han rats at dose levels of 1000, 3000 and 10000 ppm. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 40-55 days). Chemical analysis showed that the diets were prepared accurately and were homogenous.

Parental results: Parental toxicity was evident for males at females at 10000 ppm and was characterized by lower body weights and/or weight gains and treatment related macroscopic findings including enlarged liver and thyroid (seen only for a single male but considered relevant). Relative liver weights were 29% (males) and 37% (females) higher than controls. This correlated to hepatocellular hypertrophy which was observed at an increased incidence and severity during the microscopic evaluation. Higher relative thyroid weights of 50% (males) and 17% (females) were also seen at 10000 ppm; these correlated to follicular cell hypertrophy of the thyroid gland in males only. The hepatocellular hypertrophy was recorded at increased incidence and/or severity but was not accompanied by other test item-related microscopic liver findings. Additionally, the increased incidence and severity of follicular cell hypertrophy of the thyroid may reflect an increase in thyroxin production in response to feedback mechanisms as a result of increased turnover of thyroxin by hypertrophic hepatocytes. While both of these findings could be considered adaptive changes, taken together with the magnitude of organ weight increases and in the absence of clinical pathology data to provide evidence of normal functioning of these organ systems, the microscopic findings were considered to be toxicologically relevant.

The increase in relative kidney weights seen for males at 10000 ppm was only slight (7%). At the microscopic evaluation, a dose-dependent increase in the incidence and/or severity of hyaline droplet accumulation was seen for all treated males. The hyaline droplet accumulation is considered to represent alpha2uglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. This is a male-rat specific protein that is not seen in female rats or in higher mammals, including man. Taken together with the absence of other morphological hallmarks of kidney damage, the kidney effects were not considered to be adverse. No toxicologically relevant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance and food consumption).

Reproductive results:

No reproductive toxicity was observed up to the highest dose level tested (10000 ppm).

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (10000 ppm).

In conclusion, treatment with NECTARYL by diet in male and female Wistar Han rats at dose levels of 1000, 3000 and 10000 ppm revealed parental toxicity at 10000 ppm. No reproductive or developmental toxicity was observed with treatment up to 10000 ppm.

Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 3000 ppm was derived and a reproductive and developmental NOAEL of at least 10000 ppm was determined.

When corrected for mean test article intake, the parental NOAEL of 3000 ppm corresponds to 208-214 mg/kg for males and 235-348 mg/kg for females. The reproductive and developmental NOAEL of 10000 ppm corresponds to 743-750 mg/kg for males and 720-1226 mg/kg for females.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
720 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
GLP and guideline study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Reproduction/developmental toxicity screening test of NECTARYL in rats by dietary administration was performed in 2015 according to the OECD Guideline No. 421.

No reproductive or developmental effects were observed up to the highest dose tested. PArental toxicity was observed at the highest dose tested.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the data available and key results described in this summary, the substance has shown no reproductive or developmental toxicity potential and should therefore not be classified as toxic to reproduction according to the (EC) No 1272/2008 Regulation (CLP).

Additional information

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