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Diss Factsheets

Administrative data

Endpoint:
repeated dose toxicity: oral, other
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
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
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. 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 oral 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.
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).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
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.

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.

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.

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
Sacrifice and pathology:
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 from 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.
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.

Results and discussion

Results of examinations

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.
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.
All other males and females at all dose levels survived until the scheduled necropsy.
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:
effects observed, treatment-related
Description (incidence and severity):
In female No.97, group 2, euthanized in extremis on lactation day 8 due to a poor clinical condition the absolute White Blood Cell (WBC), lymphocytes (LYM), monocytes (MON) and granulocytes (GRA) counts were notably increased. This finding was associated with some gross observations including marked multiple granulomas in the lung, granulomatous inflammation of the extraocular muscles, and hyperplasia of lymphoid tissue in the ileum. Since no apparent cause of these finding was determined, a treatment-related effect cannot be excluded.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the 100 and 1000 mg/kg body weight/day group males, the value of Albumin/Globulin ratio was lower (p<0.05) compared with the control group. That was associated with a slight increase in the level of the globulin fraction (9% in a high dose group, p<0.05). A decrease in total bilirubin as compared with control values was observed in 300 mg/kg body weight/day dose male group (↓22%, p<0.05) and in 1000 mg/kg body weight/day dose females (↓26%, not significantly).
Additionally, in 1000 mg/kg body weight/day dose males a statistically significant lower level of ALP was registered when compared to the control group with a t-test (14%, p<0.05). The mean level ALP was also reduced (41%) in a high dose female group, however, without statistical significance.
These findings were correlated with hepatocellular necrosis in the 100 an 1000 mg/kg bw/day dose groups and considered as test item-related and adverse.

In 1000 mg/kg body weight/day dose recovery females, the mean serum ALT was increased (↑19%, p<0.05) and sodium ion level was decreased (↓1%, p<0.05) when compared to the control group with a t-test. Although these findings were observed only in a post-treatment period, they can be associated with histological observations in liver and kidneys, which were found in 1000 mg/kg body weight/day group and consequently considered to be treatment-related.

In two euthanized in extremis females, there were alterations in serum chemistry parameters correlated with a microscopic finding in the liver. In female No.97 (group 2), a markedly low level of bilirubin was registered (0.8 umol/L), that was associated with focal hepatocellular necrosis. In female No.135 (group 4), the markedly increased level of glutamate dehydrogenase (GLDG, 427 U/L) was associated with the generalized hemorrhagic hepatocellular necrosis.
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
Description (incidence and severity):
Organ weight difference which was considered to be associated with the administration of the test item is the decrease in relative weight of thyroids, revealed in males after the 28-days treatment period of the 1000 mg/kg body weight/day dose (15.6%, relative to body weight, and 21.6% relative to brain weight, p<0.05 as compare with t-test). The mean absolute value of thyroids weight was reduced in comparison with the control group (15.3%), however, without the statistical significance of the change. This finding was associated with histopathologic findings (slight decrease in colloid content in follicles). (No test item-related effects on T4 level)

Changes of assumed relationship to test item administration were noted in satellite males in liver, kidney, and thymus. Significant differences (p<0.05) were observed after the 1000 mg/kg body weight/day dose post-treatment as compared with vehicle control group in the following organ weights: increase in absolute and relative weights of liver (approximately 9%), increase in weight of kidneys (relative to brain weight, 7.5%), and decrease in weight of thymus (absolute and relative to body weight, approximately 22%). Increase in liver and kidneys weights was slight and was not associated with histological or/and clinical pathology changes in 1000 mg/kg body weight/day satellite males subgroups.

In some females in all test item groups, focal epithelial hyperplasia and cortex atrophy in thymus was observed.
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) and one male (scheduled euthanized) in the 1000 mg/kg body weight/day group.
Some males and 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 single male in the 1000 mg/kg body weight/day dose group and only 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.

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. 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 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).


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.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Observed effects in 100 mg/kg bw/day group were limited to one female with general poor clinical conditions. Similar effects were observed in 1000 mg/kg bw/day group. Therefore 300 mg/kg bw/day is considered as NOAEL
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: No clear relationship to test-item
Key result
Dose descriptor:
LOEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Minimal effects in 100 mg/kg bw/day groups also in control group. Additional effects at 300 mg/kg bw/day in only two males. Other effects not until 1000 mg/kg bw/day and with unclear adversity.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Neurobehavioral toxicity

Target system / organ toxicity

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

Applicant's summary and conclusion

Conclusions:
Results were reevaluated by the registrant and conclusion different to the study report is derived.
Observed effects in the thyroid gland of parental animals are not convincing. Therefore the effects on liver were used to assess repeated dose toxicity.
As observed effects on the liver in 100 mg/kg bw/day group were limited to one female with general poor condition and similiar effects were not observed until 1000 mg/kg bw/day a NOAEL instead of a LOAEL was set. A NOAEL of 300 mg/kg bw/day was adopted.
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.

No effects on FOB parameters and no effects on locomotor activity were noted at any dose level.

Hepatocellular necrosis finding in the 100 and 1000 mg/kg body weight/day dose groups, correlated to the slight increase in the level of the globulin fraction (in the 1000 mg/kg body weight/dose males), decrease in total bilirubin in 300 mg/kg body weight/day dose male group and in 1000 mg/kg body weight/day dose females, and decreased level of ALP in a high dose male and female group considered test item-related and adverse. Hepatocellular hypertrophy and accumulation of hepatocellular glycogen, associated with higher liver weight (males) and increased ALT (females) in 1000 mg/kg body weight/day satellite animals, are considered adaptive and hence non-adverse. The test item-related renal hyaline nephropathy observed in the 100, 300, and 1000 mg/kg body weight/day male and female groups 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.

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.

Therefore, under the conditions of this screening study, the no-observed-adverse-effect-level (NOAEL) was 1000 mg/kg body weight/day for F0 neurobehavioral toxicity (males and females), the lowest-observed-adverse-effect-level (LOAEL) for F0 males and pregnant and lactating female systemic toxicity was 100 mg/kg body weight/day.