[No public or meaningful name is available]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20 November 2017 to 19 June 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:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Amides, C16-18 (even numbered), N-C16-18 (even numbered) alkyl

Molecular formula:

R1-NH-C=O-R2, with R1=C16H33 and C18H37 & R2 = C15H31 and C17H35

IUPAC Name:

Amides, C16-18 (even numbered), N-C16-18 (even numbered) alkyl

Test material form:

solid

Details on test material:

- Chemical registery number : 955-212-4
- Chemical name: Amides, C16-18 (even numbered), N-C16-18 (even numbered) alkyl

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agreed by each registrant.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation: 78 to 84 days old
- Weight at study initiation: Preliminary phase: 180 to 207 g. Main phase: 182 to 225 g.
- Housing: Cages comprised of a polycarbonate body with a stainless
steel mesh lid; changed at appropriate intervals
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet ad libitum
- Water (e.g. ad libitum): Potable water from the public supply ad libitum
- Acclimation period: Six days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not
recirculated
- Photoperiod (hrs dark / hrs light): 12 hours light: 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % w/v methylcellulose and 0.1% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The required amount of test material was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder which had been wetted with vehicle, the mortar was rinsed with vehicle and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer. The suspension was transferred to the final containers, via syringe whilst magnetically stirring.

A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.

DIET PREPARATION
- Frequency of preparation: Weekly
- Storage temperature of food: Refrigerated (2 to 8°C)

VEHICLE
- Vehicle: 0.5 % w/v methylcellulose and 0.1% Tween 80
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 110 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.

Achieved concentration: Samples of each formulation prepared for administration in the first and last weeks of both the Preliminary and Main phases of treatment were analyzed for achieved concentration of the test item.

Details on mating procedure:

Male/female ratio: 1:1 with identified stock males.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation: When positive evidence of mating was detected.
A colony of stud (stock) males from the same source was maintained specifically for the purpose of mating. These animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

From Day 5 to Day 19 (inclusive) after mating

Frequency of treatment:

Once daily at approximately the same time each day.

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Preliminary study: 6 pregnant females per dose
Main study: 14 pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the results of a 14 day preliminary study conducted in Han Wistar rats, in which three group of three males and three females were treated at 250, 500 and 1000 mg/kg/day. Overall mean body weight gains and food consumption were slightly reduced for animals treated at 500 or 1000 mg/kg/day. There were no clinical signs considered related to treatment and macroscopic examination did not reveal any treatement releated lesions.

At doses of 1000 mg/kg/day, the extent of the body weight gain/food consumtion reductions were slight; and, in the absence of a clear effect of treatment, doses of 100, 330 and 1000 mg/kg/day were selected for hte preliminary phase of this study.

There were no adverse effects of treatment in the pregnant females and no clear treatment-related effects observed in their litters at does up to 1000 mg/kg/day in the preliminary phase. One out of six females, at 1000 mg/kg/day showed low body weidht gain and food consumption with a litter of ten smaller fetuses, although a relationship to treatment was uncertain and was not considered to reclude the use of 1000 mg/kg/day for the main phase. Therefore for the main phase, the dose levels remained the same as the preliminary phase at 100, 330, and 1000 mg/kg/day.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily at approximately the same time each day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily observations were made pre-dose, 1 to 2 hours after completion of dosing, and as late as possible each working day. A detailed physical examination was performed on each animal on Days 0, 4, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: Each adult was recorded on Days 0, 3 and 5-20 after mating.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the period days 0-4, 5-8, 9-13, 14-17 and 18-19 after mating inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice Day 20 after mating
- Organs examined: All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Ovaries and uterine content:

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

The number of uterine implantations sites were checked after staining with ammonium sulphide on non-pregnant animals and for apparently empty uterine horns.

Fetal examinations:

- External examinations: Yes: [Examination of nominally 50% of fetuses in each litter)]
- Soft tissue examinations: Yes
- Skeletal examinations: Yes

Statistics:

The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons the F1 approximate test was applied.

If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment data, Kruskal-Wallis’ test was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests were made. For all other comparisons the H1 approximate test was applied.

For litter size and survival indices and fetal, placental and litter weight and gravid uterine weight data, if 75% of the data (across all groups) were the same value, Fisher’s exact tests were performed using pairwise comparisons of each dose group against the control.
Pre/post implantation loss and sex ratio were analyzed by generalized mixed linear model with binomial errors, a logit link function and litter as a random effect. Each treated group was compared to control using a Wald chi-square test. For resorptions, each treated group was compared to control by exact Wilcoxon rank sum test.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Historical control data:

See attached document

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Preliminary Phase: There were no clinical signs and no signs seen in association with dosing.
Main Phase: Signs associated with dosing were observed exclusively in females treated at 1000 mg/kg/day and included, piloerection, hypoactivity (recorded as underactive), chin rubbing and pale skin color. These dosing signs were present on Days 14 after mating and, generally persisted, with increased incidence, to Day 19 after mating. Clinical signs generally supported the signs seen in association with dosing, but also included hunched posture.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Preliminary Phase: There were no unscheduled deaths.
Main Phase: Five animals were euthanized due to poor clinical condition, four of which were dispatched prior to GD 20.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Preliminary phase: Overall mean body weight gain (GD 5 to 20) was low (73% of the concurrent control), for females receiving 1000 mg/kg/day largely because the body weight gain from commencement of treatment (GD5) to GD13 was significantly low (61% of the concurrent control). The mean body weight, adjusted for gravid uterine weight, was significantly low (89% of the concurrent control) on GD20 in females receiving 1000 mg/kg/day, with significantly low adjusted body weight gain from GD 5 to 20 (20% of the concurrent control). Mean bodyweight was slightly less on GD 12 (224 g) than GD 11 (223 g) for females receiving 1000 mg/kg/day.

Main phase: Overall mean body weight gain was low (65% of concurrent control) for females receiving 1000 mg/kg/day during the treatment period (GD 5 to 20). From commencement of treatment to GD 13, group mean body weight gains for females receiving 1000 or 330 mg/kg/day were significantly low (31 or 83% of concurrent control, respectively). Thereafter (GD 13 to 20), body weight gains remained low (81 or 84% of the concurrent control, respectively) for females receiving 1000 or 330 mg/kg/day. The mean adjusted body weight was significantly low (88% of the concurrent control) on GD 20 in females receiving 1000 mg/kg/day with –4% of adjusted body weight gain relative to the concurrent control from GD 5 to 20. Mean body weight was slightly less on GD 10 (219 g) than GD 9 (222 g) for females receiving 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Preliminary phase: Mean food consumption was slightly low from commencement of treatment to termination for females at 1000 mg/kg/day. At 100 or 330 mg/kg/day, food consumption was, generally, similar to Control.

Main phase: Similarly to the preliminary phase, mean food consumption was low from commencement of treatment to termination for females at 1000 mg/kg/day. However, at 100 or 330 mg/kg/day, food consumption was also marginally lower than controls throughout the treatment period.

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:

effects observed, treatment-related

Description (incidence and severity):

Preliminary phase: For adult females treated with the test item there were no macroscopic findings at scheduled termination on GD 20.

Main phase: For adult females surviving to scheduled termination on GD 20, treatment with the test item did not reveal macroscopic findings that could be considered related to treatment. The findings were limited to animals that were euthanized prior to scheduled termination due to poor clinical condition. The signs were, in general, related to abnormal contents/dehydration of the GI tract.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Animal No. 72, receiving 1000 mg/kg/day, was euthanized due to poor clinical condition on Day 17 after mating. Between days 15 and 17 after mating, this animal showed hypoactivity, hunched posture, piloerection, and had reduced body tone, with a yellow staining of the ventral surface. No food was consumed on Days 9 to 13 after mating and progressive loss of 64g of body weight was recorded from Day 9 after mating. Macroscopic examination revealed abnormal pale contents of the stomach, fecal impaction and pale rectum, small liver and kidney weights, yellow/ pale yellow contents in the ileum or caecum and dark, enlarged adrenals. The animal was non pregnant.

Animal No. 73, receiving 1000 mg/kg/day, was euthanized due to poor clinical condition on GD 10. The animal showed hunched posture, slight hypoactivity and piloerection on GD 10. The animal did not show any clinical signs or signs associated with dosing on previous administration days. Progressive weight loss of 31g was recorded from GD 8 to 10. Macroscopic examination revealed reduced, dehydrated contents in the caecum, reduced content of the ileum, abnormal (orange) colored contents in the jejunum and no fecal pellets in the rectum.

Animal No. 74, receiving 1000 mg/kg/day, was euthanized due to poor clinical condition on GD 20. Between GD 17 to 20, this animal showed hypoactivity, hunched posture and piloerection; additionally, a sign associated with dosing observations included pallor skin color on GD 19. Food intake was low on GD 5 to 17 compared with pre-dose intake.
Macroscopic examination did not reveal any significant findings. As this animal was pregnant and reached GD 20, it was considered suitable and included in assessment of fetuses in this study.

Animal No. 75, receiving 1000 mg/kg/day, was euthanized due to poor clinical condition on GD 15. This animal showed hypoactivity, hunched posture and piloerection on Day 15 after mating. Only one gram of food per day was consumed on average from 9 to 13 after mating and progressive weight loss of 47g was recorded from Day 8 to 15 after mating. Macroscopic examination revealed abnormal contents in the stomach (pale and dehydrated), pale material in the colon, yellowish fluid in the caecum and small amounts of adipose tissue. The animal was non pregnant.

Animal No. 79, receiving 1000 mg/kg/day, was euthanized due to poor clinical condition on GD 18. Between GD 16 and 18, this animal showed hypoactivity, hunched posture, piloerection with a generally thin build and loss of hair on the forelimbs. Only 2 grams of food per day was consumed on average during GD 14 to 17 and progressive weight loss of 26 g was recorded from GD 13 to 17. Macroscopic examination revealed several findings at necropsy relating to the abnormal contents within the gastrointestinal tract (stomach, ileum, caecum, rectum) and fecal impaction in the rectum. Additionally, this animal was seen with abnormally small spleen, liver and kidneys, pale spleen and liver, and enlarged adrenals.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No abortion was reported.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Percentage of pre/post implantation loss was comparable across groups.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

one female receiving 100 mg/kg/day.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Means for number of early or late resorptions was comparable across groups.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Preliminary phase: All animals were pregnant, reproductive assessment was therefore based on six animals at 0, 100, 330 or 1000 mg/kg/day. Embryo-fetal survival, live litter size and sex ratio were unaffected by treatment.

Main phase: Two control females (1F 11, 1F 13), one female receiving 100 mg/kg/day (2F 39 - complete resorption), two females receiving 330 mg/kg/day (3F 52, 3F 54) and two females receiving 1000 mg/kg/day (4F 72 and 4F 75; both euthanized prior to GD20 for welfare reasons) were found with no live fetuses at necropsy. Considering that two additional females receiving 1000 mg/kg/day were euthanized prior to GD 20, reproductive assessment was based on 12, 13 12 and 10 females at 0, 100, 330 or 1000 mg/kg/day.

Other effects:

not examined

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

330 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Preliminary phase: There were no effects of treatment on placental, litter or fetal weights.

Main phase: There were statistically significant decreases in fetal body weights for males and females in the 1000 mg/kg/day dose group compared to the concurrent control. However, fetal body weights were unaffected by treatment at 100 or 330 mg/kg/day. Placental weights were unaffected by treatment with the test item at all dose levels. There were statistically significant decreases in fetal body weights for the male, female and combined sexes of the combined preliminary and main phases between vehicle control and 1000 mg/kg/day dose groups. This was considered test item related due to the dose response and corresponding effect on maternal body weight changes.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Embryo-fetal survival was unaffected by treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was comparable across groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was unaffected by treatment.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment.
When compared to Control, there was a slightly increased incidence of misaligned costal cartilage in the low dose (100 mg/kg/day) group that marginally exceeded the HCD range. This was not considered test item related due to lack of dose response.

Visceral malformations:

no effects observed

Description (incidence and severity):

Pathological examination of the fetuses did not reveal any visceral abnormalities
considered to be related to treatment.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

330 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the maternal and embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) of the test item was considered to be 330 mg/kg/day. The maternal NOAEL was considered to be 330 mg/kg/day based on the mortality and low body weight gains in rats receiving 1000 mg/kg/day. The embryo-fetal NOAEL, although no effect on embryo-development was established, was considered to be 330 mg/kg/day - being dependent on, and secondary to, maternal toxicity.

Executive summary:

The objective of this study was to assess potential maternal and/or developmental effects of the test item, in pregnant Han Wistar rats when administered via oral gavage during the organogenesis and fetal growth phases. The test item in vehicle was administered daily from Day 5 to Day 19 after mating at dose levels of 0, 100, 330 or 1000 mg/kg/day. The adults were euthanized and examined macroscopically on Day 20 after mating and all fetuses were removed from the uterus, killed, weighed individually and externally examined. Subsequently, half of the fetuses were examined for soft tissue abnormalities and the remaining fetuses were examined for skeletal development and abnormalities. Data were evaluated from 18, 19, 18 and 16 adult females and their litters at 0, 100, 330 and 1000 mg/kg/day, respectively.
No treatment related post dose or clinical signs were observed among the 6 females per group in the preliminary phase of the study. However, in the main phase, where the group size was larger (14 females per group), some females receiving 1000 mg/kg/day showed post dose/clinical signs of piloerection, hypoactivity and hunched posture together with significantly lower body weight and/or low food consumption: three pregnant females and two females subsequently found to be non pregnant did not tolerate treatment and were euthanized before the scheduled termination due to poor clinical conditions.
The following evaluations were based on the results of combined preliminary and main phases, unless stated otherwise.
The test item induced maternal toxicity when administered during the embryo and fetal developmental period.
Mean body weight gains during GD 5 to 13 were markedly low at 1000 mg/kg/day (40% of Controls), partly as a result of the body weight losses (in either GD 11 to 12 in the preliminary phase or GD 9 to 10 in the main phase); and were low at 330 mg/kg/day (77% of Controls). Thereafter, body weight gains improved for females at 330 mg/kg/day and improved slightly at 1000 mg/kg/day. Gravid uterine weights were comparable across groups. The lower body weight adjusted gains (GD 5 to 20) were not attributed to differences in gravid uterine weights.
Mean food consumption was lower in all treated groups, however statistical significance was achieved at 330 and 1000 mg/kg/day, only.
The findings seen at macroscopic examination of females on GD 20 were confined predominantly to animals euthanized due to poor clinical condition. Those findings consisted of abnormal contents within the digestive tract and comments associated with dehydration, but also included abnormalities in the adrenals, kidneys and liver of two animals (Animals 4F 72 and 79).
There were no effects on pregnancy and uterine parameters. No significant differences were noted and means for number of corpora lutea, implantations, early or late resorptions, percentage of pre/post implantation loss and sex ratio of the fetuses were comparable across groups.
There was no effect on embryo-fetal survival at any dose level as numbers of live foetuses closely matched the numbers of implantations. Fetal weights at 1000 mg/kg/day were marginally lower than expected, given the litter size, which was considered secondary to the maternal toxicity and not direct developmental toxicity. Despite the poor body weight performance of adult females at 1000 mg/kg/day, there was no effect of treatment on placental weights.
Pathological examination of the fetuses did not reveal any visceral or skeletal abnormalities considered to be related to treatment.
Treatment with the test item resulted in maternal toxicity at 1000 mg/kg/day, which resulted in the reduction in fetal body weights. Although embryo-fetal survival and development were unaffected by treatment at 1000 mg/kg/day, this dose was not tolerated by 3 pregnant females, and therefore the No-observed-adverse-effect-level (NOAEL) for embryo-fetal effects will be set at the highest maternally tolerated dose, 330 mg/kg/day.