Amides, C12-18, N-[3-(dimethylamino)propyl]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2010-10-20 to 2011-07-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to guidelines and GLP conform study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

deviations were considered not to affect the purpose or integrity of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl] C 12-C18 alkylamide

EC Number:

932-121-8

Cas Number:

1147459-12-8

Molecular formula:

UVCB substance not applicable

IUPAC Name:

N-[3-(dimethylamino)propyl] C 12-C18 alkylamide

Test material form:

solid

Details on test material:

- Physical state: Cream coloured solid block
- Chemical name : Alkyl amidopropyl dimethyl amine
- Lot/batch No.: 1100314301
- Expiration date of the lot/batch: 10 May 2011
- Storage condition of test material: At room temperature, light protected
- CoA:
Activity (MW = 292), 98.9%
Free Fatty Acid (MW = 208), 1.0%
Dimethylaminopropyl Amine , 260 ppm
Color, Gardner 2.5
Chain length distribution = coco pattern: ≤C8: 6.0%; C10: 6.3%, C12: 52.5%, C14: 15.7% , C16: 8.9%, ≥C18: 9.4%
Other: Label: (FULL COCO CUT) LOT NO: 1100311401 30.03.10

Specific details on test material used for the study:

- Stability under test conditions: 15 % loss of initial concentration of the low dose concentration after storage at +4 °C, therefore the formulations were prepared daily

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: (P) x approximately twelve weeks old
- Weight at study initiation: (P) Males: 293-352 g; Females: 191-221 g
- Fasting period before study: no
- Housing: in groups of five in solid floor polypropylene cages with stainless steel mesh lids. During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesch lids and softwood flakes.
- Diet (e.g. ad libitum): a pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories UK Ltd, Oxon, UK), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: for thirteen days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): 55±15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 2010-11-16 to 2011-05-18

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility of the test item
- Concentration in vehicle: 0, 1.25, 3.75, 11.3 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg/day

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of fourteen days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): individually during the period of gestation and lactation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Coco-DMAPA in the test item formulations was determined by gas chromatography (GC) using an external standard technique. The test item formulations were diluted with acetone to give a final, theoretical test item concentration of approximately 0.1 mg/mL. Standard solutions of test item were prepared in acetone at a nominal concentration of 0.1 mg/mL containing the equivalent amount of vehicle as the relevant sample. The test item formulations were sampled and analysed initially and then after storage at approximately +4°C in the dark for ten days. The test item formulations were sampled and analysed within two days of preparation.

Duration of treatment / exposure:

14 days pre-mating + 14 days mating + gestation (optional extended exposure for males): a maximum of 54 days

Frequency of treatment:

daily

Details on study schedule:

- Age at mating of the mated animals in the study: 14 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 5, 15, 45 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results from the fourteen day preliminary study there clinical, body weight, food consumption, water consumtion and necropsy findings were observed especially at 100 mg/kg/day dose and somtimes at 50 mg/kg/day dose, the dose levels for the present study were selected as 5, 15 and 45 mg/kg/day.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: see clinical observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups, live births, weight gain, clinical condition of offspring from birth to Day 5 post partum

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 43.
- Maternal animals: All surviving animals on day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day
26 post coitum.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the attachment [Histopathology] were prepared for microscopic examination.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in the attachment [Histopathology] were prepared for microscopic examination.

Statistics:

Data for males and females prior to pairing, and functional performance test data, where appropriate, quantitative data were analysed by the ProvantisTM Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Bartletts's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levenels test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test. Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers. Probability values (P) were calculated as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P≥0.05 (not significant)

Reproductive indices:

Mating Index (%) = (Number of animals mated/Number of animals paired)x100
Pregnancy Index (%) = (Number of pregnant females/Number of animals mated)x100
Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females)x100

Offspring viability indices:

Live Birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born)x100
Viability Index (%) = (Number of offspring alive on Day 4/Number of offspring alive on Day 1)x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

increased salivation in animals treated with 45 mg/kg/day from Day 1 (males) and Day 7 (females) onwards.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two mortalities occurred at the highest dose level (day 7 and day 21); however, in the absence of corroborative gross pathological and histopathological findings these deaths are likely to have been incidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced body weight in females at 45 mg/kg/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced body weight in females at 45 mg/kg/day

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Erosion or ulceration of the forestomach and hyperplasia/hyperkeratosis in the forestomach was evident in two males and two females treated with 45 mg/kg/day.

Other effects:

not specified

Description (incidence and severity):

Test substance intake: gavage

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Episodes of increased salivation were evident in animals of either sex treated with 45 rnglkglday from Day 1 (males) and Day 7 (females) onwards. An isolated incident of noisy respiration was also evident in one male treated with 45 mg/kg/day on Day 4. At 15 mg/kg/day, increased salivation was evident in one male on Days 16 and 37 and in one female on Day 17. Isolated observations of this nature are commonly observed following oral administration of an unpalatable test item formulation and in the absence of any associated changes at this dose level is not considered to be attributable to systemic toxicity. The female treated with 15 mg/kg/day that was killed in extremis had a swollen anogenital region between Days 18 and 20. This observation was due to a physical injury occurring whilst the female was paired with its respective male partner and was considered not to be related to test item toxicity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Although statistical analysis did not reveal any significant intergroup differences, females treated with 45 mg/kg/day showed an initial insult to the test item with reductions in body weight gain, and four females showed actual body weight losses during the first week of treatment. Recovery was evident thereafter. No adverse effect on food consumption or food efficiency was detected for males during the treatment period or for females during the pre-mating, gestation or lactation phases of the study.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
no data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no treatment-related effects on mating performance or fertility for treated animals when compared to controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment related effects were detected in the organ weights measured.

GROSS PATHOLOGY (PARENTAL ANIMALS)
One male treated with 45 mg/kg/day had a raised limiting ridge in the stomach. The female treated with 5 mg/kg/day that was non pregnant had a fluid filled uterus. The female treated with 15 mg/kg/day that was killed in extremis had a swollen vagina and a fluid filled uterus

HISTOPATHOLOGY (PARENTAL ANIMALS)
Erosion or ulceration of the forestomach and hyperplasia/hyperkeratosis in the forestomach was evident in two males and two females treated with 45 mg/kg/day.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

for (local) maternal toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

for reproductive / developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Details on results (F1)

VIABILITY (OFFSPRING)
No significat differences were detected for litter viability for treated animals when compared to controls.

CLINICAL SIGNS (OFFSPRING)
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls.

BODY WEIGHT (OFFSPRING)
There were no significant differences in offspring weights.

SEXUAL MATURATION (OFFSPRING)
No data

ORGAN WEIGHTS (OFFSPRING)
No data

GROSS PATHOLOGY (OFFSPRING)
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring.

HISTOPATHOLOGY (OFFSPRING)
No data

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

45 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No treatment related effects were detected in the reproductive parameters examined therefore the 'No Observed Effect Level' (NOEL) for reproductiveldevelopmental toxicity was considered to be 45 mg/kg/day.

Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test" (adopted 22 March 1996) and US EPA Health Effects Test Guidelines OPPTS 870.3650, July 2000. This study was also designed to be compatible with the Commission Regulation (EC) No 44012008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 190712006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

The test item was administered by gavage to three groups, each of ten male and ten female Wistar HanTM:RccHanTM:WISsTt rain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 5, 15 and 45 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Corn oil). Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 postpartum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post parturn. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no unscheduled deaths that were related to treatment. Episodes of increased salivation were evident in animals of either sex treated with 45 mg/kg/day throughout the treatment period. An isolated incident of noisy respiration was also evident in one male treated with 45 mg/kg/day on Day 4. No toxicologically significant effects were detected in animals of either sex treated with 15 or 5 mg/kg/day. There were no treatment-related changes in the behavioural parameters measured, no toxicologically significant changes in functional performance and no treatment-related changes in sensory reactivity. Females treated with 45 mg/kg/day showed a reduction in body weight gain during the premating period, with four females showing actual body weight losses during the first week of treatment whereas this was the case for two animals of the control group. However, this difference was not statistically significant, nor was there any difference in body weight visible during the remainder of the study. No body weight effects were detected in males treated with 45 mg/kg/day or animals of either sex treated with 15 or 5 mg/kg/day. No adverse effect on food or water consumption or food efficiency was detected.

There were no treatment-related effects on mating or conception rates for treated animals. There were no differences in gestation lengths. The distribution for treated females was comparable to controls. Of the litters born, litter size at birth and subsequently on Days 1 and 4 postpartum were comparable to controls. Offspring bodyweight gain and litter weights at birth and subsequently on Days 1 and 4 postpartum were comparable to controls. The 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was therefore considered to be 15 mg/kg/day. No treatment related effects were detected in the reproductive parameters examined therefore the 'No Observed Effect Level' (NOEL) for reproductive/developmental toxicity was considered to be 45 mg/kg/day.