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Administrative data

Endpoint:
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)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
GLP compliance:
yes
Limit test:
no

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
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
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.
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.
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.
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.
Frequency of treatment:
once daily
All animals were dosed at approximately the same time each day (09:30 – 13:15).
Duration of test:
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

Maternal examinations:
All rats were observed twice daily, once in the morning and once in the afternoon, for morbidity and mortality.
Each F0 female was also observed for signs of toxicity approximately 1 hour following dose administration.
Tthe 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 females on lactation day 13.
Locomotor activity was measured. Each animal was tested separately in a randomized order for 6-minute period.
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.
Blood samples were collected for hematology, coagulation, serum chemistry and T4 Assay
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.
The number ob implatation sites was recorded.
Ovaries and uterine content:
Organ weight from ovaries and uterus in all groups.
Microscopic examinations of ovaries an uterus from six randomly selected females of control and high dose group. Ovaries additionally in low, mid-dose and satellite groups.
Fetal examinations:
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.
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.
Indices:
Female Mating Index, %
Female Fertility Index, %
Gestation index (%, N/N)
Viability index (N/N, %) of pups

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse treatment-related clinical findings in 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.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Description (incidence and severity):
There were no test item-related effects on a thyroxin (T4) level in serum of parental males. Mean thyroxin concentrations in the dose groups did not significantly differ from the values in the vehicle control group after 28 days of test item administration as well as after the 2-weeks of post-treatment period.
Urinalysis findings:
not examined
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:
no effects observed
Description (incidence and severity):
There were no significant changes in the absolute and relative organ weights in all dose treated females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related gross findings during necropsy were observed in liver (area of dark-red discoloration in the caudal liver lobe) of one female (euthanized in extremis) in the 1000 mg/kg body weight/day group.
Some females in the 300 and 1000 mg/kg body weight/day dose groups had an area or pinpoint dark-red discoloration of the thymus. In one female in the 300 mg/kg body weight/day dose group this gross observation was correlated with microscopic finding (macrophages with dark inclusions/pigments).
Female No.97 in the 100 mg/kg body weight/day dose group was euthanized on lactation day 8 due to a poor clinical condition. Necropsy revealed the gray discoloration of lungs and dark-red discoloration of the reduced thymus.
Another female (No.103) from 100 mg/kg body weight/day dose group was euthanized after prolonged labor and total litter loss. Gross observations for this female included enlarged uterus and markedly enlarged spleen, which can be caused by loss of blood during prolonged labor.
In the 300 mg/kg body weight/day dose group, one female (No.129) has reduced one ovary from the pairs without an associated change in reproductive performance.

All other macroscopic findings occurred only in the vehicle control group or at similar frequencies in the test item and control group, or were incidental, and are not supposed to be test item related.
Neuropathological findings:
not examined
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.

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).

Hepatocellular necrosis of severe and marked grade was founded in 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.

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.

Maternal developmental toxicity

Number of abortions:
no effects observed
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
Description (incidence and severity):
The number of dams with post-natal loss of offspring was slightly higher (not significantly) in the dose treated groups (1, 3, 4, and 5 in the control group, 100, 300 and 1000 mg/kg body weight/day groups, respectively).
But there were no changes in a mean number of pups born and live litter size.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day
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:
100 mg/kg bw/day
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: unclear adversity, no correlation to changes in T4 level

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The body weights at birth in test item-treated groups did not differ significantly from the values in the control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The mean number of females and males born, the percentage of males, in test item-treated groups did not differ significantly from the values in the control group.
Changes in litter size and weights:
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.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The mean number of liveborn pups and postnatal survival in the all dose-treated group did not significantly differ from the values in the control group.
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
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.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
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.
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).

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Decrease in organ weight (Thyroid gland) and T4
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other: not significant and non adverse but relationship from the test item not excluded
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Decrease in serum T4 level
Remarks on result:
other:
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
not specified
Basis for effect level:
other: Organ weight: Thyroid gland
Remarks on result:
other: As considered in study report

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified

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:

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 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.

The LOAEL for F0 reproductive toxicity and F1 neonatal toxicity was considered to be 100 mg/kg body weight/day.