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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25.02.2018 - 28.09.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Reaction mass of N-(hydroxymethyl)hexadecan-1-amide and N-(hydroxymethyl)stearamide
EC Number:
911-553-0
Molecular formula:
not applicable
IUPAC Name:
Reaction mass of N-(hydroxymethyl)hexadecan-1-amide and N-(hydroxymethyl)stearamide
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: The Lab Animals Breeding Center “Pushchino”, Branch of Institute of Bioorganic Chemistry RAS (www.spf-animals.ru). The animals were received at the age of 3 weeks in two batches (12.12.2017 and 19.12.2017).

Age: Approximately 10-12 weeks old on day of pre-mating oestrous cycle evaluation;
approximately 14-15 weeks old at the initiation of dose administration (day 1);
approximately 16-17 weeks old when paired on study day 14 (a).

Body weight at first day of dosing (MEAN ± S.D): Males: 419 ± 35 g, N = 58
Females: 248 ± 17 g, N = 58

Only females with a regular 4-5 days cycle were taken into experimental groups. Animals considered unsuitable for the study were excluded prior to group assignement.

Husbandry: Two corridors barrier-type facility (barrier zone 2 of BTL BIBC RAS) with the automatic change of day and night time (08:00-20:00 - "Day", 20:00-08:00 - "Night") and the renewal of the room air at least 12 times hourly. Husbandry practice meets the standards defined by the Directive 2010/63/EU on the protection of animals used for scientific purposes.

Environmental Conditions: Actual mean temperature ranged from 20 °C to 24 °C and mean relative humidity ranged from 30 % to 60 %.

Cages: Following group forming and until mating, all F0 females and males were housed for two animals in solid bottom polycarbonate cages (Type-4, 1425 cm2) with bedding. Cages are equipped with steel lids, steel separators for the food and steel label holders. All cages were provided with environmental enrichment material Lignocel Nesting Ball (JRS Germany).
For mating, males were housed alone and animals were paired for mating in the home cage of the male. After mating, dams were housed alone to deliver and their litters were housed in these cages until euthanasia. Female that failed to deliver was housed individually until 55 days of dosing.
Males and females of satellite subgroup (not mated) were housed 2-3 animals per cage.

Bedding: Commercial autoclaved woodchip bedding was used (LIGNOCEL BK 8/15, JRS, GmbH).

Diet: The animals were fed Laboratory Rodent Diet (SSNIFF V1534-300 autoclavable, Spezialdiaeten, GmbH) ad libitum.

Water: Filtered tap water was provided ad libitum in standard water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item suspended in the vehicle was administered once daily orally by gavage, via an appropriately sized stainless-steel ball-tipped dosing cannula connected with syringe. A separate cannula for each group was used.
The dosage volume for all groups was 10 mL/kg body weight.
Details on mating procedure:
The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the F0 males and females avoiding sibling mating. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of sperm following a vaginal lavage. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0 (G0). If evidence of copulation was not detected after 8 days of pairing in presence of regular oestrus cycle, the female was housed with another male for which the mating was confirmed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The validated method was used for detection of the test item concentration in vehicle formulations ranging from 10 to 100 mg/mL.
Duration of treatment / exposure:
The males were dosed during study days 1-28 (14 days prior to pairing and continuing throughout the mating period) for a total of 28 days. Males of satellite subgroup (not mated) were dosed for a total of 28 days with following two weeks recovery period.

The females were dosed during study days 1 through the day prior to euthanasia (14 days prior to pairing through lactation day 13) for a total of 49-61 days. Female that failed to deliver was dosed through the day prior to euthanasia for a total of 55 days. Females of satellite subgroup (not mated) were dosed for a total of 55 days with following two weeks recovery period.
Frequency of treatment:
once daily
All animals were dosed at approximately the same time each day (09:30 – 13:15).
Details on study schedule:
Start of analytical method validation: 25.02.2018
Date of start dosing: 05.03.2018
Mating period: 18.03.2018-31.03.2018
Last necropsy of males: 16.04.2018
Last F1 litter necropsy at lactation day 13: 06.05.2018
Last female necropsy: 14.05.2018
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
All rats were observed twice daily, once in the morning and once in the afternoon, for morbidity and mortality.
Each F0 male and female were also observed for signs of toxicity approximately 1 hour following dose administration.
In addition, the presence of findings at the time of dose administration was recorded for individual animals.
Females expected to deliver were also observed twice daily during the period of expected parturition (from the day 19 of gestation) and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.
Detailed physical examinations, body weight and food comsumption were recorded for all parental animals before first dosing and regularly on a weekly basis throughout the study.
Functional Observational Battery (FOB) and locomotor activity data were recorded for half of F0 males at the end of dosing and for half of F0 females on lactation day 13.
Locomotor activity was measured. Each animal was tested separately in a randomized order for 6-minute period.
Male body weights were recorded during group assignment, on the first day of dose administration, weekly thereafter throughout the study, and prior to the scheduled euthanasia.
Female body weights were recorded during group assignment, on the first day of dose administration, and weekly thereafter until evidence of copulation was observed. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), and at day 2 (for calculation of food consumption), day 4 and day 13 post-partum.
Urine samples were collected in all F0 males before scheduled euthanasia and in satellite subgroup males after 2 weeks post-treatment before scheduled euthanasia. Diuresis was calculated and urinalysis determinations (Appearance, Glucose, Specific gravity, Blood cells, pH, Ketones ,Protein, Volume (overnight)) were performed.
Blood samples were collected for hematology, coagulation, serum chemistry and T4 Assay
Oestrous cyclicity (parental animals):
Oestrous cycles was monitored before start of the treatment to select for the females with regular cyclicity (4-5 day cycles). Vaginal smears were also monitored daily for last week during the pre-mating period with continued monitoring into the mating period until there was evidence of mating. Vaginal smears were examined before necropsy to determine the stage of the oestrous cycle and allow correlation with histopathology of female reproductive organs.
Litter observations:
After birth, pups were examined for gross malformations, sexed, individually identified, and the numbers of stillborn and live pups were recorded.
Each litter was examined daily in the cage for survival, abnormalities and all deaths were recorded. On postnatal days 4, 7 and 13, a more detailed manual clinical examination of pups with weighting was carried out.
On PND 13, the number of nipples / areolae was counted in all males and females described as a dark focal area (with or without a nipple bud) and no distinction was made between the retention of an areola or a nipple.
The external genitalia were inspected in all males on PND 13 before necropsy.
Postmortem examinations (parental animals):
A complete necropsy was conducted on all F0 animals euthanized in extremis or at scheduled termination. Necropsy included examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera. Organ weights were collected and tissues were preserved.
Tissues and organs were collected fron six randomly selected males an females and placed in 10% neutral-buffered formalin for microscopic examination.
For F0 females, the number of former implantation sites were recorded.
Microscopic examination of thyroid glands was done for all adult males and females of control and all treated groups based on the thyroxine assay data, and one male and female of day 13 pup from each litter in the control and high-dose groups.
Postmortem examinations (offspring):
Blood samples were collected from two of the surplus pups per litter euthanized during litter adjusting on PND 4, and from two of the pups per litter at termination on PND 13. Serum samples from pups of the same litter were pooled for using these “litter means” as statistical unit.
Stillborn pups, pups found dead and any pups that were euthanized in extremis were necropsied using a fresh dissection technique for macroscopic observations.
At scheduled euthanasia on PND 13, all pups from litter were euthanized and necropsied with examination for gross abnormalities and particular attention to the external reproductive genitals. One male and one female from each litter were used for thyroid evaluation (weighing).
Statistics:
All statistical tests were performed separately for each sex using Microsoft Excel (descriptive statistics) and statistical software Statistica for Window v.7.1 to compare the treated groups to the control group.
Continuous data variables were analyzed by multi-factor analysis of variance ANOVA-2, followed by the Duncan test, to determine inter-group differences. Oestrus cycle length, pre-coital intervals, gestation length, former implantation sites, clinical pathology values and FOB data were analyzed by a parametric one-way analysis of variance (ANOVA). If the results of the ANOVA were significant (p<0.05), Dunnett's test was applied to the data to compare the treated groups to the control group. The t-test was applied to compare clinical chemistry parameters in a high dose treated group and control group in the case of mean value changes. Clinical pathology values and FOB values of satellite animals were analyzed by a t-test to compare the 1000 mg/kg/day group to the control group. FOB parameters which yield scalar or descriptive data were analyzed by Fisher's Exact Test. Gamma glutamyltransferase data was subjected to the Kruskal-Wallis nonparametric ANOVA test with Dunn’s test. Male copulation, female conception, male and female mating and fertility indices of the treated groups were compared to the control group using the Chi-square test.
Pup viability and sex ratio data were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference. Mean litter weights, live litter size, and number of pups born were subjected to the parametric ANOVA test and Dunnett's test with the litter representing the experimental unit.
Histopathological findings of each treated group were compared to those of the control group by the Fisher's Exact test. Organ weights were subjected to a parametric ANOVA test and Dunnett's test. In addition, the Student t-test was used to compare the organ weights of high dose group and the control group.
Reproductive indices:
Male Mating Index, %
Female Mating Index, %
Male Fertility Index, %
Male Copulation Index, %
Female Fertility Index, %
Female Conception Index, %

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse treatment-related clinical findings in males and females, excluding three euthanized in extremis females (see Mortality).
Clinical findings noted for scheduled euthanized animals in the test substance-treated groups occurred infrequently and/or were observed in the control group, and were not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One female in the 100 mg/kg body weight/day dose group had a poor clinical condition, and a single female in the 1000 mg/kg body weight/day group was with agalactia. Clinical findings in these females were associated with microscopic observations and/or clinical pathology findings and supposed to be test item-related. Another one in the 100 mg/kg body weight/day dose group was euthanized after prolonged labor with bleeding and litter loss. Since no apparent cause of these finding was determined, a treatment-related effect cannot be excluded.
In the 100 mg/kg body weight/day group, a single male was euthanized in extremis due to poor clinical condition on day 14. The cause was determined to be trauma of esophagus and, therefore, was not attributed to test item administration.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects on mean food consumption were observed at any dosage level.
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:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No effects on FOB parameters and no effects on locomotor activity were noted at any dose level.
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 histologic findings were revealed in the liver, kidneys, thymus, and thyroids. Histologic changes with unclear relationship to test item administration were observed in the spleen.

Hepatocellular necrosis of severe and marked grade was founded in one male from the 1000 mg/kg body weight/day dose group and two females from 100 and 1000 mg/kg body weight/day groups, euthanized in extremis, correlated to the serum chemistry parameters and is considered adverse.

Slight microscopic findings in thyroids of parental animals were associated with the reduction in thyroid relative weight in 1000 mg/kg body weight/day post-treatment females and in F1 offspring (100 and 1000 mg/kg body weight/day groups, males, and 1000 mg/kg body weight/day group, females), as well as decrease in thyroxin serum level in PND 13 offspring (in the 1000 mg/kg body weight/day dose group).

The test item-related renal hyaline nephropathy is often found in rats, considered species-specific and is not toxicologically significant for humans.
Thymus epithelial hyperplasia and cortex atrophy was as incidents in 100, 300 and 1000 mg/kg body weight/day dose female groups, of minimal to slight grade, and considered non-adverse.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects on reproductive performance of males and females were observed at any dosage level.
Additionaly no test item-related effects on F0 gestation length, parturition, or reproductive organs were noted at any dosage level.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: correlated to effects in F1 thyroid gland rel. weight and serum T4 level.
Key result
Dose descriptor:
LOEL
Effect level:
300 mg/kg bw/day
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Minimal effects in 100 mg/kg bw/day groups also in control group. Addittional effects at 300 mg/kg bw/day in only two males. Other effects not until 1000 mg/kg bw/day and with unclear adversity.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
reproductive performance
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
reproductive performance
Remarks on result:
other:
Remarks:
As considered in study report

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean number of liveborn pups, live litter size and postnatal survival in the all dose-treated group did not significantly differ from the values in the control group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 300 mg/kg body weight/day dose group, the slight increase in the mean value of body weight gain was noted for males (12.8%) and females (11.2%).
These changes were not statistically significant, considered non adverse, however, the relationship from the test item not excluded.
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:
effects observed, treatment-related
Description (incidence and severity):
The statistically significant decrease in the mean serum level of thyroxin was revealed in postnatal day 13 F1 offspring in the 1000 mg/kg body weight/day dose group (approximately 14% as compared to control group). A slightly reduced thyroxin level was also observed in 300 mg/kg body weight/day dose group, but this change was not statistically significant (6.7% versus control value).
This finding was correlated with the decrease in relative weights of thyroids in 100 and 1000 mg/kg body weight/day dose F1 males (↓21.7% and ↓18.8%, p<0.05 according to t-test) and in 1000 mg/kg body weight/day F1 female group (↓22.3%, p<0.05 according to Dunnet’s test).
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The relative weights of thyroids were reduced that was statistically significant in 100 and 1000 mg/kg body weight/day dose males (21.7% and 18.8%, p<0.05 according to t-test) and in 1000 mg/kg body weight/day female group 22.3%, p<0.05 according to Dunnet’s test). The change in thyroids weight was not associated with histopathologic findings any concomitant microscopic changes in thyroid tissue in F1 offspring. However, it was correlated with reduced thyroxin level revealed in high dose treated offspring and considered to be test item-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the 1000 mg/kg body weight/day dose group, one pup necropsied as found dead had red discolorated kidney and urinary bladder associated with mark edema of the neck area. These findings were not observed in the control group and can not be excluded as potentially test item-related.

However, the visceral examination of all pups (stillborn and died) did not indicate any test item-related effects on the morphological development of the offspring.
There were no test item-related gross observations in all pups during scheduled necropsy on PND 13. No signs of demasculinization were found in any male in all groups.
Histopathological findings:
no effects observed
Description (incidence and severity):
No significant histological changes were observed in any of the thyroid glands examined in postnatal day 13 offspring in the control and 1000 mg/kg body weight/day dose group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: Observed effects in 100 mg/kg bw/day male group are of unclear adversity
Key result
Dose descriptor:
LOEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: unclear adversity
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: Correlated with decrease in T4 level in 1000 mg/kg bw/day group
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
clinical biochemistry

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects

Applicant's summary and conclusion

Conclusions:
Results were reevaluated by the registrant and conclusion different to the study report is derived.
As observed effects in parental animals in 1000 mg/kg bw/day are correlated to observed effects in 1000 mg/kg bw/day F1 a LOAEL is set to 1000 mg/kg bw/day and a NOAEL of 300 mg/kg bw/day is assumed.
Executive summary:

No body weight loss, no decrease in body weight gain and no reduced food consumption in males was observed during 4 weeks and in females up to lactation day 14 at the dose levels of 100, 300 and 1000 mg/kg body weight/day. There were no adverse treatment-related clinical findings in males and females, excluding three euthanized in extremis females. One female in the 100 mg/kg body weight/day dose group had a poor clinical condition, and a single female in the 1000 mg/kg body weight/day group was with agalactia. Clinical findings in these females were associated with microscopic observations and/or clinical pathology findings and supposed to be test item-related. Another one in the 100 mg/kg body weight/day dose group was euthanized after prolonged labor with bleeding and litter loss. Since no apparent cause of these finding was determined, a treatment-related effect cannot be excluded.

Slight microscopic findings in thyroids of parental animals were associated with the reduction in thyroid relative weight in 1000 mg/kg body weight/day males and in F1 offspring (100 and 1000 mg/kg body weight/day groups, males, and 1000 mg/kg body weight/day group, females), as well as decrease in thyroxin serum level in PND 13 offspring (in the 1000 mg/kg body weight/day dose group).

No test item-related effects on F0 reproductive performance, gestation length, parturition, or reproductive organs were noted at any dosage level. Also, there were no changes in a mean number of pups born, live litter size, postnatal survival, physical conditions and any signs of offspring masculinization or feminization. The slight increase in the mean value of body weight gain was noted for F1 offspring in the 300 mg/kg body weight/day dose group. In PND13 F1 offspring, the decrease in relative weights of thyroids was observed in 100 and 1000 mg/kg body weight/day dose, and a reduction in serum level of thyroxin was observed in the 1000 mg/kg body weight/day dose group.

Therefore, under the conditions of this screening study, the LOAEL for F0 reproductive toxicity and F1 neonatal toxicity was considered to be 100 mg/kg body weight/day.