Fatty acids, C16-18 (even numbered), ammonium salts

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 2017 - 26 March 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 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Fatty acids, C16-18 (even numbered) ammonium salts

IUPAC Name:

Fatty acids, C16-18 (even numbered) ammonium salts

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Cmdb: WI; outbred

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Medicine Centre at the Medical University in Białystok
- Age at study initiation: 12 weeks (range finding ), 13 weeks (main test)
- Weight at study initiation: 229.2 g (range finding), 246.3 g (main test). Body weights were within ±20% of the average value.
- Housing: At mating one female was placed in one cage with one male. At pregnancy the females were housed individually in plastic cages with metal wire lid and dimensions: 58 x 37 x 21 cm (length x width x height).
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 3 weeks (range finding), 2 weeks (main test).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 41-80
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

VEHICLE
- Amount of vehicle (if gavage): 0.5 mL /100 g b.w.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples of the test item dispersions in distilled water of each dose from four time points during the main study were chemically analyzed. Samples were collected at the beginning of the experiment, two times during the main study and at the end- at the last day of dosing animals of all three dose levels. The samples were analysed by a validated HPLC coupled with UV-DAD method.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 1 day (range finding), 1-3 days (main test)
- Verification of same strain and source of both sexes: Pregnant females were obtained by mating with not related males.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

From 5th to 19th day of gestation

Frequency of treatment:

Once a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

33.1 mg/kg bw/day (actual dose received)

Remarks:

Group 1

Doses / concentrations 2

Dose / conc.:

181.8 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 3

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A range finding study was performed.
Doses: 0 (control), 57.2, 229 and 916 mg/kg bw/day.
Number of females per group: 7.
Methodology: The same as in the main test.
On the basis of DRF study results doses for the main study were selected. No significant maternal toxic effects occurred in the highest dose of DRF study (except volvulus, which was not necessarily caused by the test item action), slight changes as decrease in food consumption between the 5th and 11th day of gestation occurred. Changes in fetuses were a decrease of weight of fetuses with placenta and fetal membranes and a decrease of weight of placenta itself. Therefore, the highest dose for the main study was maintained at similar level as 1000 mg/kg b.w. Since at the lowest dose changes as increased weight of placenta and decreased length of fetuses (with and without tail) occurred and the lowest dose should not produce evidence of toxicity, the lowest dose for the main study was decreased to 33.1 mg/kg b.w. The middle dose was 181.8 mg/kg b.w. to obtain a separation factor of 5.5.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day during labour week and once a day on days off.
- Parameters: Observation for morbidity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Day o and then 5th, 8th, 11th, 14th, 17th and 20th day of gestation.
- Parameters: Skin changes, coat changes, changes in eyes and mucous membranes,
respiratory system, circulatory system, nervous system, somatic activity and behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: on 0, 2nd, 5th, 8th, 11th, 14th, 17th and 20th day of gestation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Controlled on days of their weighing
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 of gestation.
- Organs examined:

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (formation of particular cavities and presence of internal organs was evaluated)
- Skeletal examinations: Yes: half per litter (skull bones, back-bone, ribs, acromial and pelvic girdles with limbs was evaluated. Points of ossification in sternum, metacarpus of fore limbs and metatarsus of hind limbs were counted during evaluation).
- Head examinations: Yes: all per litter
- Other: Sex of each fetus, body weight, length with tail and without tail was determined. Weight of the fetuses with placenta and fetal membranes was determined first and then weight of the fetuses without placenta and fetal membranes as well as weight of placenta were measured. During the gross evaluation attention was paid to: reactions to tactile impulses, formation of body coverings, formation of limbs, number of fingers and toes, shape of head, formation of auricular conchas, presence of nasal apertures, formation of oral cavity and presence of anus and tail.

Statistics:

The obtained results were elaborated statistically with the use of STATISTICA 10, with p ≤ 0.05. The results obtained in the treated groups (group 1, group 2, group 3) were compared with the control group (group 0). For each quantitative parameter, the basic characteristics of descriptive statistics were determined: mean, standard deviation, variance and using the Shapiro-Wilk test, it was assessed whether a given parameter had a normal distribution. Then, the following statistical tests were used to assess intergroup relationships:
• for quantitative parameters having normal distribution: a one-way analysis of variance, preceded by Bartlett's test, checking the homogeneity of variance, and then a post-hoc test: Dunnett's test.
• for quantitative parameters having a non-normal distribution or in the case of heterogeneity of variance: Kruskal-Wallis test, if necessary confirmed with Dunnett’s test.

Indices:

Body weight of pregnant females, food consumption of pregnant females, number of corpora lutea, number of fetuses in litter, number of males and females in litter, weight of uteri, weight of fetuses, length of fetuses, number of ossification points in sternum and limbs.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Single cases of coat thinning (in one female per dose in each treatment group) and alopecia (in one female in group 1) occurred only in treated groups. The frequency of this change did not increase with the increase of the dose. Furthermore, the single cases of coat thinning or alopecia were observed in control groups of other studies performed in the laboratory, so it should not be connected with the test item.
The influence of the test item on treated animals was observed at the highest dose. Twelve cases of transient respiratory murmurs were observed in group 3 and there were no similar changes in lower doses and in the control group.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female of group 3 died at day 9. All remaining females in the main study survived the entire period of the experiment. Clinical changes as distinct decrease of locomotor activity, dejection, bristled coat and difficult respiration, which were observed in this female before death were not observed in the lower doses and in the control group and seem to be connected with the test item. All these changes in the highest dose of 1000.0 mg/kg b.w. could be seen as the influence of the test item on rat females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the entire period of the experiment average body weight of the females of group 1 and group 2 did not differ statistically significantly from average body weight of the females of the control group. The average body weight of the females of group 3 was statistically significantly lower in comparison with the control group in 14th day of gestation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the experiment, food consumption of the females in group 1 and group 2 was not statistically significantly different from the control group. Food consumption of the females in group 3 was statistically significantly lower in comparison with the control group from 5th to 20th gestation day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No pathological changes were stated at gross examination of the pregnant females from group 0,1, 2 and 3 performed at caesarean section.
In three females found out to be non-pregnant (1 in control group, 1 in group 1 and 1 in group 2) the presence of clear fluid in uterus was stated, which is normal during proestrus phase of estrous cycle in female rats. In one non-pregnant female from group 1 the presence of a follicular cyst in the ovary was stated, what could be the cause of ineffective mating of this female, due to the possibility of hormonal disturbances affecting ovulation and hence fertility.
Post- mortem examiniation of one female from group 3, which died in 9th day of the experiment and was not pregnant revealed following findings:
- lungs enlarged, with emphysema and foci of congestion
- digestive tract filled with gas,
- congestion of intestines, liver and kidneys.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

Fertility was equal in all groups, including control group, 23 pregnant females per group were obtained.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Resorptions were observed in all experimental groups. Intrauterine mortality was comparable in the low and the high dose with the control group and was three times lower in the mid dose than in the control group.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Mortality of 3 fetuses in group 2 (where the resorption number was the lowest) and 1 fetus in group 3 is not high, especially since one of the dead fetuses from group 2 was found to be wrapped around with the umbilical cord and his death cannot be regarded as caused by the test item.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The average weight of the uterus of pregnant females of the treated groups did not differ statistically significantly from average weight of the uterus of pregnant females of the control group.

Details on maternal toxic effects:

The average number of corpora lutea in all treated groups are comparable with the number of corpora lutea in the control group.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

181.8 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase of weight of fetuses with and without placenta and fetal membranes in treated groups together with the increase of length of fetuses in treated groups (statistically significant in group 2- mid dose) can indicate the test item as inducing the intensive growth of fetuses. This was accompanied by an increase in the number of ossification points in sternum and metacarpus (discussed further). Although an increase of weight in treated versus control group is statistically significant, based on historical and literature data fetuses of all treated groups are within the range of normal body weight of 20 day rat fetuses

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

Variations in sternum ossification centres were of unilateral, bipartite and misaligned character and occurred in all treated groups and in control group. Moreover the incidence of variations in sternum ossification centres in group 1 were comparable with the control group and the occurence of changes was decreasing with the increase of the dose, that allouds to conclude that the test item did not have adverse effect on the process of the ossification of the sternum. Other stated lesions like lack of cranial vault and wavy ribs occurred only in control group without causative effect of the test item.
Analysis of percentage of fetuses with particular number of ossification points of sternum revealed approximately 40% of fetuses with 5 and 6 ossification points in control group and almost 18% of fetuses with 4 ossification points. One can clearly see that in treated groups percentage of fetuses with 6 ossification points increased to over 50% (in group 1 almost 55%, in group 2 the value reached nearly 70% and in group 3 over 60%) and number of fetuses with 5 and 4 ossification points decreased. Statistical analysis of the number of ossification points in sternum confirmed that the average value of ossification points was statistically significantly greater in all treated groups (at low, mid and high dose). This observations lead to the conclusion that the test item influenced on
acceleration of the sternum ossification process.
Analyzing percentage of fetuses with particular number of ossification points in metacarpus, clearly visible is an increase in percentage of fetuses with 4 ossification points and a decrease in percentage of fetuses with 3 ossification points along with the increasing dose of the test item. Statistical analysis of the number of ossification points in metacarpus confirmed that the average value of ossification points was statistically significantly greater in all treated groups and that ossification of metacarpus progressed faster along with increasing doses. Above facts indicate the test item as accelerating ossification of metacarpus of forelimbs. There is no significant difference in ossification of metatarsus of hindlimbs between treated and control groups, because in all groups percentage of fetuses with 4 points of ossification is nearly (control group and group 3) or exactly 100% (group 1 and 2).

Visceral malformations:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The average lengths of the fetuses with tail and lengths of the fetuses without tail were statistically significantly greater in group 2 compared with the control group.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: Not adverse effects observed at the highest dose tested.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Results of mating:

Parameter	Number of females
	Group 0	Group 1	Group 2	Group 3
No. of females	25	25	25	25
No. of pregnants	23	23	23	23
No. of deaths	-	-	-	1
No. of not pregnants	2	2	2	2
Females with resorptions	8	10	4	7

Clinical signs:

Parameter	Number of females
	Group 0	Group 1	Group 2	Group 3
No. of females	25	25	25	25
Coat thinning on back and alopecia on side	-	1	-	-
Coat thinning on forelimbs	-	-	1	-
Coat thinning on abdomen and limbs	-	-	-	1
Respiratory murmurs	-	-	-	12
Distinct decrease of locomotor activity, dejection, bristled coat and difficult respiration	-	-	-	1
Body weight loss – pregnant females	1	-	5	13
Body weight loss – not pregnant females	2	2	2	2

Body weights:

Days of gestation	Number of females
	Group 0	Group 1	Group 2	Group 3
No. of females	23	23	23	23
0	245.22 ± 20.33	247.30 ± 16.02	245.74 ± 13.95	245.65 ± 16.97
5	262.65 ± 23.19	266.83 ± 17.19	265.52 ± 15.44	263.48 ± 17.83
8	271.70 ± 24.06	274.04 ± 17.60	269.70 ± 17.46	260.39 ± 20.55
11	284.52 ± 26.45	289.96 ± 18.61	283.57 ± 17.77	270.22 ± 20.25
14	296.04 ± 26.92	301.65 ± 18.61	295.17 ± 18.79	280.35 ± 18.83*
17	321.35 ± 31.16	329.57 ± 19.43	322.78 ± 20.87	308.35 ± 19.31
20	357.70 ± 37.03	370.83 ± 24.05	359.04 ± 24.66	348.00 ± 20.15

* statistically significant difference with p ≤ 0.05

Food consumption:

Days of gestation	Number of females
	Group 0	Group 1	Group 2	Group 3
No. of females	23	23	23	23
0-5	7.66 ± 0.56	7.81 ± 0.51	7.81 ± 0.56	7.56 ± 0.81
5-8	7.85 ± 0.80	7.87 ± 0.56	7.61 ± 0.55	6.69 ± 1.18*
8-11	7.47 ± 1.00	7.60 ± 0.96	7.21 ± 0.84	6.38 ± 1.13*
11-14	7.92 ± 0.65	7.71 ± 0.69	7.61 ± 0.78	6.56 ± 0.95*
14-17	7.51 ± 0.56	7.33 ± 0.52	7.25 ± 0.36	6.80 ± 0.82*
17-20	6.95 ± 0.57	6.78 ± 0.49	6.73 ± 0.59	6.30 ± 0.71*

* statistically significant difference with p ≤ 0.05

Applicant's summary and conclusion

Conclusions:

The NOAEL for maternal toxicity was determined to be 181.8 mg/kg bw/day based on decrease of food consumption and decrease of body weight gain, transient respiratory murmurs and a problable death of one female rat. The test item did not cause adverse effects in fetuses, therefore the NOAEL for fetal toxicity was determined to be 1000 mg/kg bw/day.

Executive summary:

A prenatal developmental toxicity was performed according to the OECD Guideline 414 (GLP study). After a dose range finding, the main test was conducted on 100 females divided into four experimental groups: three treated groups and one control group. Each group consisted of 25 females. From day 5 to 19 of gestation the test item dispersed in distilled water was administered to the females of each group by gavage at 0 (distilled water only), 33.1, 181.8 mg/kg and 1000 mg/kg bw/day. During the study, clinical observations for mortality and signs of toxic influence of the test item were performed in females, their body weight and food consumption were controlled. On day before an expected delivery, on day 20 of gestation, all females were killed with the use of morbital and caesarean section with gross examination. The following endpoints were determined: number of alive and dead fetuses, number of resorptions. After the removing of fetuses each uterus was weighed. The non-gravid uteri and non-gravid horns were overexposed by electric lamp and were stained using ammonium sulphide in order to confirm the non-pregnant status. In the main study the number of corpora lutea was determined in fixed ovaries. The following endpoints were determined in all fetuses: sex, body weight with and without placenta, weight of placenta, body length with and without tail. Each fetus was subjected to gross examination. Half of fetuses from each litter were stained with alizarin and subjected to evaluation of skeleton. The rest of them were evaluated for formation of body cavities and internal organs. Based on the study and analysis of the results it can be stated that the test item did not cause clinical changes and changes in behavior of pregnant females in group 1 and group 2. In group 3 the test item affected respiratory system (what manifested itself by transient respiratory murmurs in twelve females) and probably caused death of one female and clinical symptoms which preceded the death of this female. Additionally, the test item at the highest dose affected decrease of food consumption and decrease of body weight gain in females. Since the statistically significant lower food consumption and lower body weight, clinical signs and death case in group 3 seems to be caused by the test item, the NOAEL for maternal toxicity can be defined as 181.8 mg/kg b.w. Greater average body weights of fetuses in all treated groups and the increase of length of fetuses in group 2 and increases in the average number of ossification points of the sternum and the metacarpus in the treated groups seem to be caused by the test item. However, they are no adverse effects, as they do not diminish an organism’s ability to survive, reproduce or adapt to the environment. Therefore, on the grounds of the used doses, the NOAEL for fetal toxicity can be defined as 1000 mg/kg b.w.