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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No full animal studies are available on effects on fertility. No indications for reproduction toxicity are indicated by results form available 28-day, 90-day andCombined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening studiesonCoco amidopropyldimethylamine. Possible exposures are very limited due to the characteristics and use of the substance.

Link to relevant study records

Reference

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

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

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650

GLP compliance:

yes

Limit test:

no

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

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

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

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.

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

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: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

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.

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

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

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

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

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

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

45 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OneCombined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening studyonCoco amidopropyldimethylaminewas recorded for this endpoint and identified as a key study.

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) following OECD Guidelines for Testing of Chemicals No. 422.

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han 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 5 post 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 toxicologically significant effects detected in the haematological or in the blood chemical parameters measured. No treatment related effects were detected in the organ weights measured. One male treated with 45 mg/kg/day had a raised limiting ridge in the stomach. No toxicologically significant effects were detected in females treated with 45 mg/kg/day or in animals of either sex treated with 15 or 5 mg/kg/day. Basically, the only toxicologically relevant finding was the observation of slight to marked findings of erosion/uceration and hyperplasia/hyperkeratosis in the forestomach which is considered a consequence of local corrosive/irritating properties of the dosed formulation.

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 45 mg/kg/day as the few effects observed at this dose-level are considered local in nature, to be caused by the corrosive/irritant properties of the dosed formulation. 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.

 

No adverse effects on reproductive organs or functions were identified in the combined repeated dose toxicity study (OECD 422) or the two available repeated dose toxicity studies (OECD 407 & 408) with Coco amidopropyldimethylamine up to 60 mg/kg bw/day. There were also no effects on pairing, mating, fertility parameters, pups survival or body weight at this same dose-level. A developmental toxicity study performed on Coco amidopropyldimethylamine also included endpoints relevant to assessing an effect on fertility. No effects on pre/post implantation rate, late/early resorptions, corpora lutea or number of live foetuses were seen in this study.

Thus, in accordance with Section 8.7.3 of column 1, Annex IX of REACH, no further Extended One-Generation Reproductive Toxicity Study is therefore indicated.

 

 

There is no repeated dose toxicity study via inhalation route available. Cocoamidopropyldimethylamine is a solid with a melting point of 23°C that is marketed or used in a non-solid or non-granular form and with a vapour pressure around 3.96 10-5 Pa at 25°C (EPI suite estimation) far below 0.1 Pa. Also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be unlikely to occur.

 

The substance is corrosive for skin. Therefore, dermal route is not the preferred route for reproduction toxicity studies.

 

In addition, there is no consumer exposure toCocoamidopropyldimethylamine.Manufacture and use are highly controlled. Its use is limited to industrial and professional users where following its severe corrosive properties sufficient protection measures will be in place to prevent exposure. Furthermore, Cocoamidopropyldimethylamine are not expected to easily pass the skin, and in view of their severe corrosive properties, testing via dermal route for reproduction toxicity is not a first choice.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity (OECD 414, GLP) is available on Cocoamidopropyldimethylamine: NOAEL for embryo-foetal toxicity was found to be 100 mg/ kg body weight, the highest dose tested.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 September 2016 - 29 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 3 november 2015

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen
- Solubility and stability of the test substance in the solvent/vehicle: stable as solution in corn oil for at least 5 hours at room temperature and 11 days in the refrigerator (confirmed over the concentration range 1 to 200 mg/mL)

Species:

rat

Strain:

other: Crl:WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks.
- Weight at study initiation: 174-231 g
- Fasting period before study: none
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm). Sterilized sawdust as bedding material and paper as cage enrichment/nesting material were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 - 21.2
- Humidity (%): 50.1 – 74.8
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 September 2016 (first delivery of mated females) To: 29 September 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item (0.885 g/cm3 at 20 °C) and vehicle (0.92 g/cm3). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch
- Amount of vehicle (if gavage): 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (12 September 2016), according to a validated method (LC-MS/MS, validated in a range 1-250 mg/g). Samples of
formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Details on mating procedure:

Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

From Days 6 to 20 post-coitum, inclusive

Frequency of treatment:

Once daily, 7 days/week

Duration of test:

14 days

Dose / conc.:

0 mg/kg bw/day

Remarks:

concurrent vehicle controls

Dose / conc.:

15 mg/kg bw/day

Dose / conc.:

40 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose:

22 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 28-day repeated dose study (OECD 407), with a 7-day dose range finder in advance, and a combined 28-day repro screening study (OECD 422), with a 14-day dose range finder in advance.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily for clinical signs from day 2 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced, as no treatment related effect was suspected

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: ovaries and uterine horns

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]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter]

Statistics:

The following statistical methods were used to analyze the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
• Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

For each litter the following calculations were performed:
Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites)/number of corpora lutea) x 100%
Post-implantation loss (%) = ((number of implantation sites - number of live fetuses)/number of implantation sites) x 100%
Viable fetuses affected/litter (%) = ((number of viable fetuses affected/litter) / (number of viable fetuses/litter)) x 100%

Historical control data:

Historical control data from the same testing laboratories in 2014-2015 are available (see under "Any other information on results incl. tables').

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Rales and piloerection were noted at 100 mg/kg bw/day for five and one female(s), respectively. Although these signs recovered after 1-5 days, they might be related to treatment with test item.
Salivation was noted for all females at 100 mg/kg bw/day, compared to none in the other groups. This finding was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Moreover, alopecia was noted for one single female at 15 mg/kg bwd/day, which was not considered to be treatment related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Treatment at 100 mg/kg bw/day resulted in two unscheduled deaths.
One female at 100 mg/kg bw/day was sacrificed in extremis on Day 7 post-coitum (before dosing), the day after having received the first dose. Observations preceding early sacrifice included squeaking, rales, labored (severe) and shallow (moderate) respiration, gasping and hypothermia. At necropsy, dark red foci were noted on the thymus. This female was pregnant and had 8 normal implantations in development.
Another female of the 100 mg/kg bw/day group was found dead on Day 21 post-coitum, the day of planned necropsy. No toxicologically relevant clinical signs were noted for this female. Body weight and food consumption was slightly lower for this female, when compared to the other females of the 100 mg/kg bw/day group. This was considered to be (partly) caused by the number of fetuses, as she had only 3 (dead) fetuses and 4 early resorptions. No macroscopic alterations were noted at necropsy.
Although these mortality findings were incidental, it could not be excluded that these unscheduled deaths were related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a trend towards lower body weights at 100 mg/kg bw/day from Day 12 post-coitum onwards. Body weight gain was statistically significantly lower at 100 mg/kg bw/day than controls on Days 15 and 18 post-coitum. For uterus corrected body weight gain was considered to be unaffected by treatment up to 100 mg/kg bw/day.
Mean body weight and body weight gain at 15 and 40 mg/kg bw/day remained in the same range as controls.
One female from the control group and three females from the 100 mg/kg bw/day group had significantly lower body weights and weight gains when compared to the other females of the same group. This was considered to be (partly) due to the relatively low number of fetuses, as two females had 1 fetus, and two other females had 3 fetuses. In addition, for two females (one with one fetus and one with two fetuses) no body weight gain was observed over Days 12 to 21 post-coitum, which was considered to be a direct treatment-related effect rather than only caused by the low number of fetuses.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before and after correction for body weight was statistically significantly reduced at 100 mg/kg bw/day on Days 9 to 12 post-coitum. This recovered during the remainder of the treatment period.
At 15 and 40 mg/kg bw/day, food consumption before or after correction for body weight were similar as controls.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

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, non-treatment-related

Description (incidence and severity):

Alopecia was noted for one single female at 15 mg/kg bw/day, confirming the clinical sign observed during the in-life phase.
Dark red foci on the thymus were noted for one female at 100 mg/kg bw/day, which was early sacrificed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre- or post-implantation loss were unaffected by treatment up to and including 100 mg/kg bw/day.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower numbers of corpora lutea (10.7 per female) were noted in the 100 mg/kg bw/day group when compared to the concurrent control group (12.2 per female). As treatment started from implantation onwards, i.e. Day 6 post-coitum, this was not considered to be treatment related. Consequently, the number of implantation sites at 100 mg/kg bw/day were slightly lower than controls as well (9.6 versus 11.4 per female). This was not statistically significant and within the range of available historical control data and not considered to be treatment related.

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no effects on fetal body weights (both sexes) noted by treatment up to 100 mg/kg bw/day.
Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.2 and 5.4 gram for the control, 15, 40 and 100 mg/kg groups, respectively
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Mean litter sizes were 10.7, 10.7, 11.0 and 9.1 viable fetuses/litter for the control, 15, 40 and 100 mg/kg bw/day groups, respectively.
The slightly lower number of fetuses/litter at 100 mg/kg bw/day when compared to controls was not statistically significant and within the range of available historical control data. As it was related to the lower numbers of corpora lutea and implantation sites at 100 mg/kg bw/day, it was not considered to be treatment related.
There were two females, one in the control group and one in the 100 mg/kg bw/day group, which only had one viable fetus. This was considered to be chance findings.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 100 mg/kg bw/day.
Mean sex ratios (males:females) were 55:45, 49:51, 43:57 and 50:50 for the control, 15, 40 and 100 mg/kg groups, respectively.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.7, 10.7, 11.0 and 9.1 viable fetuses/litter for the control, 15, 40 and 100 mg/kg groups, respectively.
The slightly lower number of fetuses/litter at 100 mg/kg bw/day when compared to controls was not statistically significant and within the range of available historical control data. As it was related to the lower numbers of corpora lutea and implantation sites at 100 mg/kg bw/day, it was not considered to be treatment related.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to 100 mg/kg bw/day.
External malformations occurred in all groups. A small lower jaw was observed in one fetus of 100 mg/kg bw/day group and was skeletally confirmed. An absent tail combined with anal atresia were found in one fetus of 40 mg/kg bw/day group, and in 15 mg/kg bw/day group an omphalocele in one fetus and anasarca in another fetus were noticed. Omphalocele and anasarca were observed in single control fetuses as well. The single occurrence and/or group distribution of the above malformations did not indicate a treatment relationship and therefore all were considered to be chance findings.
External variations were not seen in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment up to 100 mg/kg bw/day.
Malformations were observed in two fetuses of 100 mg/kg bw/day group and single fetuses of control, 15 and 40 mg/kg bw/day groups. A fetus from the 100 mg/kg bw/day group (which had a small lower jaw and a variety of visceral malformations) appeared to have severely malaligned sternebrae and a bent scapula as well. Another fetus of the 100 mg/kg bw/day group had malpositioned metatarsals.
The other affected fetuses (control, 15 and 40 mg/kg bw/day groups, respectively) all had a vertebral anomaly with or without associated rib anomaly. In addition, the fetus from the 15 mg/kg bw/day group (that also had an omphalocele and malpositioned kidneys) had a sternal anomaly and bent limb bones as well. The group distribution and/or single occurrence of these malformations did not suggest any treatment relationship and therefore all were considered to be chance findings.
Skeletal variations occurred at an incidence of 86.7%, 89.3%, 78.5% and 76.0% per litter in controls, 15, 40 and 100 mg/kg bw/day, respectively. All the ones noted, were not considered treatment-related, as they occurred in the absence of a dose-dependent relationship, infrequently and/or at frequencies that were within the range of available historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 100 mg/kg bw/day.
In total five fetuses were viscerally malformed in this study. The two fetuses of 15 mg/kg bw/day group, one fetus of the 100 mg/kg bw/day group and one control fetus that were affected externally, appeared to have one or more visceral malformations as well. The other affected fetus was a 40 mg/kg bw/day group fetus. Due to the group distribution and variety of malformation observed, all were considered not to be toxicologically relevant.
Visceral variations were observed in 10.3%, 9.1%, 7.3% and 7.5% of fetuses per litter in controls, 15, 40 and 100 mg/kg bw/day groups, respectively. These all occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects observed at the highest tested dose.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Analytical verification of the tested formulations

The concentrations analyzed in the formulations were in agreement with the target concentrations (mean accuracies 100.9% (n = 6), 98.4 (n = 2) and 99.3 (n = 6) for 15, 40 and 100 mg/kg bw/day dose solutions).

The formulations of low- and high-dose groups were homogeneous ( coefficient of variation 0.6 and 1.0, respectively (n = 6)).

Summary of developmental effects observed in the study

Dose level (mg/kg bw/day)	0	8	25	75
Pregnant/total dams	22/22	22/22	22/22	22/22
-early resorptions -late resorptions (% per litter)	7.1 0.4	5.5 0.0	6.2 0.3	4.6 0.0
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses only	0	0	0	0
Pre-implantation loss (number and percent)	19 (6.2%)	26 (9.3%)	26 (9.1%)	21 (10.7%)
Post-implantation loss (number and percent)	14 (7.5%)	14 (5.5%)	19 (6.5%)	10 (4.6%)
Body weight on day 21	327	326	326	313
Body weight gain day 6-21 (%)	48	47	47	43
Gravid uterine weight (g)	76.5	76.2	76.4	65.3
Mean live offspring (number)	10.7	10.7	11.0	9.1
Live offspring (percent)	92.5	94.5	93.5	95.4
Mean fetal/pup body weight males (g)	5.5	5.5	5.3	5.5
Mean fetal body weight females	5.1	5.2	5.1	5.2
Mean fetal body weight (sexes combined)	5.3	5.3	5.2	5.4
Malformations (including runts) number and percent of fetuses per litter	3 (1.4%)	2 (0.9%)	3 (2.3%)	2 (1.5%)
Variations (% per litter) -external -soft tissue -skeletal	0 10.3 86.7	0 9.1 89.3	0 7.3 78.5	0 7.5 76.0

Historical control data (2014-2015)

Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)
Gestation Day 21		Mean of Study Means
Study Date Range: 2014 - 2015
Endpoint	Total	Mean	SD	Median	Min	Max	P5	P95
No of Studies	13
Total No. of Animals in the Control Group	304
No. of Animals that Died	0
No. of Animals that were Euthanized	0
No. of Animals that Aborted or Delivered	3
Percent Pregnant		98.8	2.73	100.0	90.9	100.0	97.1	100.0
No. of Animals Examined at Laparohysterectomy	301
No. Nongravid	4
No. Gravid	297
No. with Only Resorptions	2
No. of Dams with Live Fetuses	295
Mean No. Viable Fetuses/Dam		10.7	0.71	10.6	9.1	11.6	10.3	11.2
Total No. Viable Fetuses	3194
Viable Fetuses (%/Litter)		95.2	2.63	95.9	88.9	98.4	93.6	96.8
Mean No. Postimplantation Loss/Dam		0.5	0.15	0.4	0.2	0.7	0.4	0.6
Total No. Postimplantation Losses	134
Postimplantation Loss (%/Litter)		4.8	2.63	4.1	1.6	11.1	3.2	6.4
Dead Fetuses (%/Litter)		0.0	0.11	0.0	0.0	0.4	0.0	0.1
Early Resorptions (%/Litter)		4.7	2.62	4.1	1.6	11.1	3.2	6.3
Late Resorptions (%/Litter)		0.0	0.11	0.0	0.0	0.4	0.0	0.1
Mean No. Implantations/Dam		11.2	0.69	11.1	9.6	12.0	10.8	11.6
Mean No. Corpora Lutea/Dam		11.9	0.71	11.7	10.9	13.2	11.5	12.3
Mean No. Preimplantation Loss/Dam		0.7	0.32	0.6	0.2	1.3	0.5	0.9
Total No. Preimplantation Losses	207
Preimplantation Loss (%/Litter)		6.2	3.43	5.8	2.0	14.5	4.2	8.3
Total No. Male Fetuses	1617
Total No. Female Fetuses	1577
% Males/Litter		50.8	2.12	50.7	46.6	53.7	49.5	52.0
% Female/Litter		49.2	2.12	49.3	46.3	53.4	48.0	50.5
Mean Fetal Body Weight (g)		5.2	0.08	5.2	5.1	5.3	5.1	5.2
Mean Male Body Weight (g)		5.4	0.10	5.4	5.2	5.5	5.3	5.4
Mean Female Body Weight (g)		5.1	0.07	5.1	5.0	5.2	5.0	5.1
Mean Male Placenta Weight (g)^1		0.46	0.01	0.47	0.44	0.47	0.4	0.5
Mean Female Placenta Weight (g)^1		0.44	0.01	0.44	0.42	0.45	0.4	0.5

 

¹Based on 4 datasets

Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)
Gestation Day 21
Study Date Range: 2014 - 2015
No. of Studies	13
Total No. Fetuses/Litters Examined Externally	3194	295
Total No. Fetuses/Litters Examined Viscerally	2061	295
Total No. Fetuses/Litters Examined Skeletally	2059	295
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
MALFORMATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
Total External Malformations								1	1
Total Visceral Malformations								7	7
Total Skeletal Malformations								15	15
Total Malformations								22	22
EXTERNAL
Exencephaly	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Eye(s)- Open	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
VISCERAL
Diaphragmatic Hernia	0.0	0.08	0.0	0.0	0.3	0.0	0.1	1	1
Eye(s)- Absent and/or Small	0.1	0.26	0.0	0.0	0.9	0.0	0.2	3	3
Hydrocephaly- External	0.0	0.12	0.0	0.0	0.5	0.0	0.1	1	1
Situs Inversus	0.2	0.34	0.0	0.0	1.0	0.0	0.4	3	3
SKELETAL
Jaw- Upper Jaw Small	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Jaw- Lower Jaw Absent or Small	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Limb Bone(s)- Bent	0.3	0.44	0.0	0.0	1.1	0.0	0.5	4	4
Rib Anomaly	0.1	0.31	0.0	0.0	1.1	0.0	0.3	1	1
Skull Anomaly	0.1	0.34	0.0	0.0	1.2	0.0	0.3	2	2
Sternebra(e)- Fused	0.1	0.29	0.0	0.0	1.0	0.0	0.3	2	2
Sternebra(e) Malaligned (Severe)	0.0	0.08	0.0	0.0	0.3	0.0	0.1	1	1
Sternoschisis	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Vertebral Anomaly With or Without Associated Rib Anomaly	0.2	0.53	0.0	0.0	1.9	0.0	0.5	3	3
Vertebral Centra Anomaly	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1

Historical Control Data Rat: Crl:WI(Han) (outbred. SPF-Quality)
Gestation Day 21
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
VARIATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
EXTERNAL
None Observed
VISCERAL
Kidney(s)- Renal Papilla(e) Absent and/or Small	0.1	0.25	0.0	0.0	0.9	0.0	0.2	2	2
Liver- Appendix	1.2	0.56	1.3	0.3	2.3	0.9	1.6	23	21
Liver- Discolored	0.1	0.30	0.0	0.0	1.0	0.0	0.3	3	3
Liver- Small Supernumerary Lobe(s)	4.0	1.96	4.0	1.3	7.7	2.8	5.2	69	58
Spleen- Supernumerary	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Thymus- Partially Undescended Horn(s)	1.3	1.55	0.8	0.0	4.3	0.3	2.2	34	23
Thyroid- Discolored	0.1	0.36	0.0	0.0	1.3	0.0	0.3	1	1
Ureter(s)- Convoluted	1.0	2.39	0.0	0.0	8.7	0.0	2.5	43	28
Ureter(s)- Dilated	0.9	2.33	0.0	0.0	8.5	0.0	2.3	44	19
SKELETAL
7th Cervical Rudimentary Rib(s)	1.7	1.34	1.2	0.0	4.4	0.9	2.5	30	26
7th Cervical Full Rib(s)	0.1	0.36	0.0	0.0	1.1	0.0	0.4	2	2
14th Full Rib(s)	5.7	4.65	5.2	0.0	13.1	2.9	8.5	88	64
14th Rudimentary Rib(s)	44.1	19.84	54.4	19.0	72.0	32.1	56.1	798	250
Metacarpal(s) and/or Metatarsal(s) Unossified	2.2	1.97	1.0	0.0	6.3	1.0	3.4	41	24
Pelvic Girdle- Caudal Shift	6.6	3.77	7.1	1.7	12.8	4.3	8.9	127	71
Rib(s)- Bent	10.6	7.78	10.2	0.8	22.3	5.9	15.3	162	85
Rib(s)- Short	0.0	0.06	0.0	0.0	0.2	0.0	0.0	1	1
Skull- Reduced Ossification	2.7	2.55	1.8	0.0	7.0	1.2	4.3	81	46
Skull- Supernumerary Site	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Sternebra(e) #1, #2, #3 and/or #4 Unossified	0.2	0.31	0.0	0.0	0.8	0.0	0.3	3	3
Sternebra(e) #5 and/or #6 Unossified	0.9	1.33	0.0	0.0	4.1	0.1	1.7	37	23
Sternebrae- Malaligned (Slight or Moderate)	11.1	5.72	8.9	4.4	21.3	7.6	14.5	188	131
Sternum- Supernumerary Ossification Site	0.1	0.31	0.0	0.0	1.1	0.0	0.3	1	1
Vertebral Centra- Reduced Ossification	0.6	0.88	0.4	0.0	3.0	0.1	1.2	12	12

Conclusions:

In a GLP-compliant guideline study with rats, the administration of the test substance by oral gavage to pregnant rats during gestation days 6-21 did not result in developmental effects on the offspring at the highest tested dose of 100 mg/kg bw/day. This level was considered to be a NOAEL for developmental effects. Based on the clinical signs and reduced body weight gain at the highest dose level in maternal animals, the NOAEL for maternal toxicity was set at 40 mg/kg bw/day.

Executive summary:

In a GLP-compliant OECD guideline 414 study, the test substance was administered to groups of 22 pregnant rats at dose levels of 0 (concurrent vehicle controls), 15, 40 and 100 mg/kg bw/day. 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. Rales and piloerection were observed in 5 and 1 female of the high-dose group, respectively, but disappeared after 1 -5 days. Lower body weight gains were observed on days 15 and 18 of gestation. Based on these effects the maternal NOAEL was set at 40 mg/kg bw/day.

Statistically significantly lower numbers of corpora lutea were noted in the high-dose group in comparison to the controls, resulting in a lower number of implantation sites; however, as the treatment started after the implantation, i.e. from day 6 post-coitum, this finding was considered to be not related to the treatment. Furthermore, the number of implantation sites was within the range of historical control data. There were no effects on the number of early and late resorption, post-implantation losses or the number of viable fetuses. Body weights and sex ratio of fetuses were unaffected by the treatment. There were no treatment-related external, visceral or skeletal variations or

malformations at any dose level. Based on this, the NOAEL for developmental toxicity was set at the highest tested dose of 100 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

NOAEL developmental toxiicty is 100 mg/kg bw/day; the NOAEL for maternal toxicity was 40 mg/kg bw/day, based on clinical signs, lower BW gains on days 15 and 18 of gestation and two incidental deaths observed at at 100 mg/kg bw/day.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available (further information necessary)

Additional information

In a GLP-compliant OECD guideline 414 study, the test substance was administered to groups of 22 pregnant rats at dose levels of 0 (concurrent vehicle controls), 15, 40 and 100 mg/kg bw/day. 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. Rales and piloerection were observed in 5 and 1 female of the high-dose group, respectively, but disappeared after 1 -5 days. Lower body weight gains were observed on days 15 and 18 of gestation. Based on these effects the maternal NOAEL was set at 40 mg/kg bw/day.

Statistically significantly lower numbers of corpora lutea were noted in the high-dose group in comparison to the controls, resulting in a lower number of implantation sites; however, as the treatment started after the implantation, i.e. from day 6 post-coitum, this finding was considered to be not related to the treatment. Furthermore, the number of implantation sites was within the range of historical control data. There were no effects on the number of early and late resorption, post-implantation losses or the number of viable foetuses. Body weights and sex ratio of foetuses were unaffected by the treatment. There were no treatment-related external, visceral or skeletal variations or malformations at any dose level. Based on this, the NOAEL for developmental toxicity was set at the highest tested dose of 100 mg/kg bw/day.

 

Cocoamidopropyldimethylamineis a solid/paste with mp of 23°C and has a vapour pressure ofaround 3.96 10-5 Pa at 25°C (EPI suite estimation) far below 0.1 Pa. Its use is limited to industrial and professional users and does not involve the forming of aerosols, particles or droplets of an inhalable size. So exposure to humans via the inhalation route will be unlikely to occur.

Manufacture and use are highly controlled. Its use is limited to industrial and professional users where following its severe corrosive properties will provide for sufficient protection measures to prevent exposure. Furthermore, Cocoamidopropyldimethylamine are not expected to easily pass the skin, and in view of their severe corrosive properties, testing via dermal route for reproduction toxicity is not a first choice.

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, reproduction toxicity is not expected. The observed effects are local, reflecting a point-of-first-contact effect.

In physiological circumstancesCocoamidopropyldimethylaminehas a cationic surfactant structure (nitrogens are fully protonated)which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar hydrophobic tails are pushed out of solution and easily dissolve in the lipid bilayer, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Cytotoxicity through disruption of cell membrane at exposure site will occur rather than absorption over the cell membrane. Consequently, significant uptake followed by placental transfer is not expected to occur.

Justification for classification or non-classification

On the basis of available studies and according to regulation 67-548 EEC and CLP regulation(EC) N° 1272/2008 the substance is not classified as a reproductive toxicant.