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Effects on fertility

Description of key information

A NOAEL of 1000 mg/kg bw was observed in male and female Sprague-Dawley rats in a reproductive/developmental toxicity screening study according to OECD guideline 421 (Calvert Laboratories, Inc., 2010; Klimisch 1). Based on the available data and according to the CLP criteria, the test substance should not be classified as toxic to reproduction.

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
other justification
Justification for data waiving:
other:
Reproductive effects observed:
not specified
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-13 - 2010-11-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Amine C8
- Physical state: dark liquid
- Storage condition of test material: at room temperature, 10 to 27 °C
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: (P) a minimum of 13 weeks old at initiation of cohabitation; records of dates of birth for animals used in this study will be retained in the Calvert archives
- Weight at study initiation: (P) Males: > or = 300 g; Females: > or = 200 g at initiation of cohabitation
- Fasting period before study: no data
- Housing: Upon arrival and until randomization, males and females are group-housed, sexes separate. Following randomization and until cohabitation, males and females will be individually housed. During cohabitation, one female will be placed with a male breeder from the same group. Following cohabitation, males and females will be housed individually. No later than gestation Day 17, mated female animals are plaec in totes with bedding. Animals will be housed in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals". The room in which the animals are kept is documented in the study records. No other species are kept in the same room
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): All animals have access to Harlan Teklad Rodent Diet (certified) or equivalent ad libitum, unless otherwise specified. No contaminants are known to be present in the certified diet at levels that would be expected to interfere with the results of this study. Analysis of the diet was limited to that performed by the manufacturer, records of which will be maintained in the Calvert archives.
- Water (e.g. ad libitum): Water will be available ad libitum, to each animal via an automatic watering device. The water routinely analyzed for contaminants as per Calvert SOP's. No contaminants are known to be present in the water at levels that would be expected to interfere with the results of this study. Results of the water analysis will be maintained in the Calvert archives.
- Acclimation period: Study animals will be acclimated to their housing for a minimum of 7 days prior to their first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26°C
- Humidity (%): 30 to 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark, except when room lights are turned on during the dark cycle to accomodate blood sampling or other study procedures

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test article and vehicle control preparations are prepared weekly or additionally as needed by diluting the test article in vehicle (w/v) to reach the proper concentrations.

Dose formulation samples:
On the first day of dosing, at the beginning of cohabitation and at the last day of dosing, duplicate 1-mL samples are obtained from top, middle, and bottom of each formulation, including the vehicle control, to determine the concentration and homogeneity of the test article in the vehicle. These samples are stored at room temperature, approximately 10 to 30°C.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Details on mating procedure:
Animals are mated by placing one male and one female from the same dose group overnight in a breeding cage until evidence of copulation is noted, after which the male animal is separated. Animals remain in cohabitation for a maximum of three weeks. During this time, the study Director may elect to move certain females with other males from the same dose group, in an attempt to expedite mating. This procedure is documented in the study raw data records.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Males: The vehicle control or test article formulations are administered once daily for a minimum of four weeks (starting two weeks prior to cohabitation). Treatment continues during the same group cohabitation period and until the day before scheduled for euthanasia. Each animal received a mL/kg dose based upon its most recent body weight.
Females: The test/vehicle control or test article formulations are administered once daily for a minimum of 15 days prior to cohabitation, during cohabitation and from presumed gestation days 0 through day 19 of gestation.
Procedure: Each animal receives a mL/kg dose based upon its most recent body weight.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control vehicle
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based upon previously conducted toxicity studyes
- Rationale for animal assignment (if not random): at random
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality: twice daily; once prior to scheduled sacrifice
- Cage side observations: each animal is observed for evidence of death or impending death (as per Calvert SOP VET-14)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: throughout the treatment phase, a minimum of twice daily, prior to dose administration and a minimum of once following dosing. On non-dosing days, a minimum of once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: males: once weekly prior to initiation of cohabitation, during cohabitation and at terminal sacrifice. A final body weight is also obtained for all males sacrificed moribund; females: once weekly prior to initiation of cohabitation on gestation days 0, 4, 7, 10, 14, 17 and 20 (day 23 and 26 if required), and on Day 0 and 4 of lactation. A final body weight is also obtained for all females showing signs of premature delivery or sacrificed moribund.

FOOD CONSUMPTION:
Males and females: frequency: full feeder weights and/or feeder weigh backs are recorded once weekly prior to cohabitation, on gestation days 0-4, 4-7, 7-10, 10-14, 14-17, 17-20 (20-23 and 23-26 if required), and on Day 0 and 4 of lactation. During cohabitation food consumption is not recorded

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

Oestrous cyclicity (parental animals):
Estrous cycle evaluation is performed daily for 2 weeks prior to cohabitation and daily during the treatment and cohabitation periods. Day 0 of gestation is determined by evidence of copulation which is determined by the examination of vaginal smears made daily to determine if sperm are present in a smear of vaginal contents or by the presence of a copulatory plug in situ. Examination of vaginal smears is performed at approximately the same time each day (early morning) throughout the cohabitation period.
Postmortem examinations (parental animals):
SACRIFICE
a) Method of euthanasia:
All adult animals are sacrificed by CO2 asphyxiation. All male animals are euthanized 2 weeks post-mating and all females are euthanized on Day 4 of lactation. All pups are euthanized by an intrathoracic injection of a barbiturate overdose.
b) Unscheduled deaths of males and females:
For all males and females sacrificed moribund or found dead prior to scheduled sacrifice, a complete gross necropsy is performed. The necropsy includes the examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. The ear tag, all gross lesions, ovaries, uterus, cervix, vagina, seminal vesicles and prostate are retained in 10% neutral buffered formalin for possible histopathological evaluation.
Additionally, for male rats, the testes and epididymides are retained in modified Davidson's fixative for possible histopathological evaluation. Where applicable, the total number of implantation sites and the total number of corpora lutea for each ovary are recorded. Where applicable, the number of viable and non-viable fetuses are also recorded. All fetuses are discarded.
c) Terminal sacrifice of males:
all surviving male animals are euthanized 2 weeks post-mating and a macroscopic examination are performed. The necropsy includes the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents.
d) Terminal sacrifice of females:
All surviving females are euthanized on Day 4 of lactation and a macroscopic examination is performed. The necropsy includes the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The total number of implantation scars are also determined.

GROSS NECROPSY
A complete gross necropsy is performed by Calvert personnel on all adult animals that are sacrificed or found dead during the study. The necropsy includes examination of:
- the external body surface
- all orifices
- the cranial, thoracic and abdonimal cavities and their contents
All abnormalities are described completely and recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
For all male animals at scheduled sacrifice at 14 days post cohabitation, the following organs are weighed before fixation, after dissection of excess of fat and other excess tissues. Organ weights are not recorded for animals found dead or sacrificed moribund: epididymides, testes. Organ to body weight ratios are calculated (using the final body weight obtained prior to necropsy), as well as organ to brain weight ratios.
Tissue collection and preservation: all tissues for all adult animals are examined. For all animals necropsied, the tissues are preserved in 10% neutral buffered formalin (except for the epididymides and testes that will be retained in modified Davidson's fixative for optimum fixation). Tissues collected: ovaries, uterus, cervix, vagina, testes, epididymides, prostate, seminal vescicles. Special emphasis on stages of spermatogenesis and histopathology of the interstitial testicular structure are given.
Postmortem examinations (offspring):
SACRIFICE
All pups are euthanized by an intrathoracic injection of a barbiturate overdose on Day 4 of lactation. All neonates are sexed, weighed, examined as soon as possible after delivery for litter seize, still births, live births and any gross anomalies. Neonates are not retained. All neonate malformations are photographed.

Statistics:
Statistical analysis will be performed on in-life and necropsy data when 3 or more animals are present in 2 or more dose groups. Statistical analysis is not performed if N<3 animals per group. For in-life parameters, the homogeneity of the data is determined by Bartlett's Test. If the data is homogeneous, a one-way analysis of variance is performed to assess statistical significance. If statistically significant differences between the meand are found, Dunnett's test is used to determine the degree of significance from the control means (p<0.05 and p<0.01). If the data is non-homogeneous, the Kruskal-Wallis non-parametric analysis is performed to assess statistical significance. If statistically significant differences between the means are fround (p<0.05, p<0.01), the Mann-Whitney U-Test is used to determine the degree of significance from the control means (p<0.05 and p<0.01). If only 2 dose groups are present for evaluation, the Mann-Whitney U-Test is used to assess statistical significance between the 2 groups. For necropsy organ weight data, the evaluation of the equality of means is made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, Dunnett's test is used to determine the degree of significance from the control means (p<0.05 and p<0.01). If only 2 dose groups are present for evaluation, the Mann-Whitney U-test is used to assess statistical significance between the 2 groups. For necropsy organ weight data, the evaluation of the equality of means is made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, Dunnett's test is used to determine the degree of significance from the control means (p<0.05 and p<0.01).
Reproductive indices:
Pre-coital interval (in days): sum of days until successful copulation/number of presumed pregnant animals
Copulation index (%): (number of presumed pregnant animals/number of paired animals) x 100
Fertility index (%): (number of pregnant animals/number of presumed pregnant animals) x 100
Preimplantation loss (%): (no. of corpora lutea - number of implantations/number of corpora lutea) x 100
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female animal was found dead on lactation day 2. All other females survived until scheduled sacrifice.
All male animals survived until scheduled sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant changes in bw or bw gain (in females and males)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No biologically relevant effects on food consumption during study (in females and males)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic toxic effects observed at 1000 mg/kg/dose (males and females)
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
no data
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
no data
Reproductive performance:
no effects observed
Description (incidence and severity):
In Life: One animal was non-gravid, another female did not mate. All males appeared normal.
Post mortem: no definitive effects on percent gravidity. No effects on pre-coital interval, copulation index and duration of gestation period were noted.
no statistically significant findings in the number of gravid animals, corpora lutea/dam, total implantations and pre-implantation loss.
no treatment-related differences in litter viability parameters.
no statistically significant changes in fetal sex ratios.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Incidence of neonates born alive/found dead, stillborn or missing between lactation days 0-4 was comparable among study groups.
One female (1000 mg/kg) was found dead on lactation day 2, and all neonates were stillborn.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment-related differences in litter viability parameters.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the neonates were statistically significantly increased. The biological significance of this finding is unknown.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related malformations were observed for neonates.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No statistically significant changes in fetal sex ratios.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
No treatment-related differences in litter viability parameters.

Neotnate Observation
Incidence of neonates born alive/found dead, stillborn or missing between lactation days 0-4 was comparable among study groups.
One female (1000 mg/kg) was found dead on lactation day 2, and all neonates were stillborn.
No treatment-related malformations were observed for neonates.
No statistically significant changes in fetal sex ratios.

BODY WEIGHT (OFFSPRING)
Neonate bw were statistically significantly increased. the biological significance of this finding is unknown.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
In a screening reproductive / development toxicity test (performed according to OECD guideline 421), rats were exposed to 1000 mg test substance/kg bw/d orally (via gavage). No adverse effects were observed. A NOAEL of 1000 mg/kg bw/d was derived. The test substance is not to classified as reproductive toxicant according to the criterial laid down in the CLP Regulation.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A Reproductive / Development Toxicity Screening Testing in Rats was performed according to OECD guideline 421 (Calvert Laboratories, Inc., 2011, Klimisch 1). In this GLP-compliant study, male and female Sprague-Dawley rats (10 animals/sex/dose) were exposed to 1000 mg/kg bw/day. Control animals received concurrent vehicle (water). The rats were exposed on a daily basis, starting two weeks prior to cohabitation until the day before the scheduled euthanasia (minimum of 4 weeks) for males and starting for a minimum of 15 days prior to cohabitation, during cohabitation and from presumed gestation days 0 through day 19 of gestation for females. One female animal was found dead on lactation day 2. All other females survived until scheduled sacrifice. No treatment-related effects were observed in clinical signs, mortality, body weight (gain), food consumption, gross pathology, organ weights or histopathology in the parental animals. There was no treatment-related effect on reproductive performance or in reproductive parameters like for instance number of gravid animals, corpora lutea per dam, total implantations, litter viability or fetal sex ratios. The incidence of neonates born alive/found dead, stillborn or missing between lactation days 0 -4 was comparable among study groups. No treatment-related malformations were observed for neonates. The body weight of neonates was statistically significantly increased with an unknown biological significance for this finding. A NOAEL of 1000 mg/kg bw/day was established.


An extended one-generation reproductive toxicity study according to OECD443 is currently being performed. This information will be submitted later based on ECHA communication/decision number CCH-D-2114352349-44-01/F

Effects on developmental toxicity

Description of key information

A prenatal development toxicity study in rabbits was performed according to OECD guideline 414 (Renaut, 2019). It was concluded that the NOEL was 250 mg/kg/d for both maternal toxicity and embryo-fetal development.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Data waiving:
other justification
Justification for data waiving:
other:
Species:
rat
Abnormalities:
not specified
Developmental effects observed:
not specified
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-07-27 - 2019-11-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Huntsman Europe BVBA and 17/19961
- Expiration date of the lot/batch: 2019-11-22
- Purity test date: Not applicable (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature (15 to 25°C); protected from light and moisture.
- Stability under storage conditions: Not specified
- Stability under test conditions: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2and 200mg/mL were analyzed to assess the stability of the test item in the liquid matrix.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Not specified
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixed to vehicle (purified water)
- Final preparation: The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.
A series of formulations at the required concentrations were prepared in ascending order.
- Frequency of preparation: Weekly, in advance of dose administration.
- Storage of formulation: Refrigerated at 2 to 8°C

OTHER SPECIFICS
- Physical State/appearance: Brown liquid
- other information: No correction factor

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS.
- Age at study initiation (day 0 of gestation): 19 to 23 weeks old
- Weight at study initiation: 2.82 to 4.61 kg
- Fasting period before study: No
- Housing: Suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. Cages were also fitted with a plastic resting platform.
Number of animals per cage: acclimatization one female, during mating one stock male and one female, during gestation one female.
- Diet (e.g. ad libitum): Teklad 2930 Diet, the diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Availability was restricted (initially 150g/animal/day during acclimatization up to one week prior to the onset of mating and 200g/animal/day thereafter). Should an individual show a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with 20 to 50 mL of water) was offered and the consumption was recorded. In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.

- Water (e.g. ad libitum): Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were offered to individuals during acclimatization.
- Acclimation period: 19 days before commencement of mating.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 21°C
- Humidity (%): 45 - 70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 10/14

IN-LIFE DATES: From: 2018-07-04 To: 2018-08-24
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
-The test item was prepared at the appropriate concentrations as a solution in Distilled Water.
- The stability of formulations was demonstrated over a period of up to 21 days at 2 to 8°C and one day at ambient temperature (15 to 25°C). Formulations were therefore prepared weekly and stored at approximately 2 to 8 ºC.
VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable (vehicle is water)
- Concentration in vehicle: 0, 25, 50,100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability was confirmed for Amine C8 in Distilled Water formulations at nominal concentrations of 2 mg/mL and 200 mg/mL when stored refrigerated for 21 days and at room temperature in the light for 24 hours.
Samples of test item formulations were taken on two occasions and analyzed for concentration and the results indicate that the prepared formulations were within 100-112% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 6 days
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: not specified
- Proof of pregnancy: Expelled uterine contents were identified and examined, as appropriate
- Any other deviations from standard protocol: No applicable
Duration of treatment / exposure:
Day 6 to 28 after mating
Frequency of treatment:
Once daily at approximately the same time each day.
Duration of test:
29 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control)
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: not specified
- Rationale for animal assignment (if not random): Where possible only females mating at least twice were allocated.
Method to group and cage position in the sequence of mating: Females mating on any one day were evenly distributed amongst the groups.
Allocation was controlled to prevent any stock male from providing more than one mated female in each treated group and to prevent more than one sibling female in each group, where possible.

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. A detailed physical examination was performed on each animal on Days 0, 6, 12, 18, 24 and 29 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded weekly during acclimatization, on Days 0, 3 and 6-29 after mating

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 1 after mating.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Fetuses (live and dead).
Fetal examinations:
- External examinations: Yes [half per litter]
- Soft tissue examinations: Yes [ half per litter]
- Skeletal examinations: Yes [half per litter]
- Head examinations: Yes: [ half per litter ]
Statistics:
The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights:

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre-treatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon1945) were made. For all other analyses the/The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs were observed at either routine physical examination or in association with dose administration that could be related to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female dosed at 125 mg/kg/day was euthanized prematurely with evidence of abortion. Uterine examination revealed a single implantation that was resorbing. This was therefore considered to be a resorption rather than abortion and as there was a solely a single implant it is considered to relate to biological efficiency rather than an effect of treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
As treatment progressed weight gain for females receiving 500 mg/kg/day was low when compared with Controls and overall the bodyweight gain from GD6 to GD29 bodyweight gain was 17% of Controls (p<0.01). The overall body weight gain for females at 125 or 250 mg/kg/day were similar to the Controls and showed no adverse effect of treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption for females receiving 500 mg/kg/day was slightly but consistently low from Day 6 to Day 27 of gestation when compared with Controls, with the total consumption over the treatment period at approximately 79% of the Control value.
At 250 mg/kg/day there were occasions of slightly low consumption however overall the food consumed was similar to Control values.
Food consumption at 125 mg/kg/day was unaffected by treatment with Amine C8.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The maternal body weight loss following adjustment for the gravid uterine weight was slightly greater than Controls for females that received 500 mg/kg/day; however this difference did not attain statistical significance. The maternal weight loss for females at 125 or 250 mg/kg/day were similar to the Controls and showed no adverse effect of treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of females did not reveal any findings that could be attributedto treatment with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
On GD22 one female dosed at 125 mg/kg/day was euthanized prematurely with evidence of abortion. Uterine examination revealed a single implantation that was resorbing. This was therefore considered to be a resorption rather than abortion and as there was a solely a single implant it is considered to be incidental and relate to biological efficiency rather than an effect of treatment.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The post-implantation loss (%) for females at 500mg/kg/day was slightly high when compared with Controls and the resultant mean litter size was low when compared with Controls (p<0.05). However, the live litter size and percentage of post implantation loss were within the historical control data range so no effect of treatment is inferred.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
On GD29 prior to sacrifice, one female dosed at 500 mg/kg/day prematurely delivered six live offspring, one dead offspring and one late resorption within the animal facility. Macroscopic examination of female no 72 and offspring did not reveal any findings that could be attributed to treatment. As this was an isolated incidence it was considered to be unrelated to administration of test item.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One control female and 3 females treated at 250 mg/kg/day were found to be not pregnant at macroscopic examination. There was no effect that could be attribute to treatment on the number of pregnant females.
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Group mean placental weights, male or fetal weight and overall fetal weight were essentially similar to Controls at dose levels up to and including 500 mg/kg/day.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
There was no clear effect of treatment on offspring survival and litter size at 125 or 250 mg/kg/day. The post-implantation loss (%) for females at 500 mg/kg/day was slightly high when compared with Controls and the resultant mean litter size was low when compared with Controls (p<0.05). However, the live litter size and percentage of post implantation loss were within the historical control data range so no effect of treatment is inferred.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
The mean litter weight for females that received 500 mg/kg/day was slightly but significantly low when compared with Controls; this was attributed to the low live litter size rather than the mean fetal weight at this dose level.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The overall incidence of minor external abnormalities was not adversely affected by test item treatment. The incidences of individual categories of minor abnormalities were within normal ranges for all groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The total number of fetuses with major abnomalities were 0, 3, 0, 4 in control, 125, 250 and 500mg/kg/day respectively. The range and incidence of major abnormalities at 125 and 500 mg/kg/day showed no relationship to treatment.
The overall incidences of minor skeletal abnormalities were similar for all groups. One statistically significant difference was observed: at 500 mg/kg/day there was a slight increased incidence of full supernumerary 13th ribs and unilateral caudal shift of pelvic girdle when compared with both the Concurrent Controls and the historical control data range. The combination of these skeletal variants at 500 mg/kg/day indicates a slight shift in rib/vertebral configuration which is not considered adverse.
Visceral malformations:
no effects observed
Description (incidence and severity):
The overall incidence of minor visceral abnormalities was not adversely affected by test item treatment. The incidences of individual categories of minor abnormalities were within normal ranges for all groups.
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
no
Relation to maternal toxicity:
not specified
Relevant for humans:
not specified

Table1: Number of pregnant dams, dams with abortions, early deliveries, resorptions, and/or dead foetuses.

Group/Sex

Total number of dams

Number of pregnant dams

Number of dams with abortions

Number of dams with early deliveries

Number of dams with resorptions

Number of dams with dead

foetuses

1F

22

21

0

0

12

0

2F

22

22

1

0

12

0

3F

22

19

0

0

6

0

4F

22

22

0

1

16

1

Table 2: Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (kg) on Day 29 of gestation

Group/Sex

 

Body weight Day 6

Terminal body weight Day 29

Body weight change 6-29

Gravid uterine weight

Adjusted body weight Day 29

Adjusted body weight change 6-29

Statistics test

 

Av

Wi

Wi

Wi

Wi

Wi

1F

Mean

3.74

3.96

0.22

0.533

3.43

-0.31

 

SD

0.247

0.237

0.163

0.1019

0.218

0.188

 

N

21

21

21

21

21

21

2F

Mean

3.77

4.04

0.26

0.504

3.53

-0.24

 

SD

0.341

0.335

0.142

0.1221

0.349

0.172

 

N

21

21

21

21

21

21

3F

Mean

3.67

3.87

0.21

0.484

3.39

-0.28

 

SD

0.325

0.345

0.156

0.0929

0.313

0.139

 

N

19

19

19

19

19

19

4F

Mean

3.76

3.83

0.07**

0.462*

3.37

-0.39

 

SD

0.310

0.266

0.212

0.1029

0.254

0.174

 

N

21

21

21

21

21

21

Table 3: Litter data - group mean values on Day 29 of gestation

Group/Sex

 

Corpora lutea

Implantations

Resorptions Early

Resorptions Late

Resorptions Total

Live young Male

Live young Female

Live young Total

Sex ratio (%M)

Pre-Implantation loss (%)

Post-Implantation loss (%)

Statistics test

 

Wi

Wi

 

 

 

 

 

Wi

Wi

Wi

Wi

1F

Mean

10.8

10.0

0.7

0.3

1.0

4.8

4.2

9.0

53.6

7.3

10.3

 

SD

2.05

2.01

0.91

0.73

1.14

2.09

2.07

2.06

19.16

7.97

11.30

 

N

21

21

21

21

21

21

21

21

21

21

21

2F

Mean

9.8

9.0

0.9

0.2

1.1

3.7

4.2

7.9

49.5

9.4

10.9

 

SD

2.02

2.50

1.28

0.89

1.45

1.15

2.04

2.22

18.41

16.32

13.15

 

N

21

21

21

21

21

21

21

21

21

21

21

3F

Mean

10.0

8.6

0.3

0.2

0.5

3.2

4.9

8.1

40.2

14.2

5.1

 

SD

1.49

1.68

0.58

0.50

0.96

1.46

1.96

1.73

17.93

12.81

10.63

 

N

19

19

19

19

19

19

19

19

19

19

19

4F

Mean

10.2

9.3

0.9

0.7

1.6

3.9

3.9

7.7*

50.0

8.5

16.2

 

SD

1.81

2.13

1.04

1.23

1.80

1.85

1.93

2.12

17.80

13.27

16.46

 

N

21

21

21

21

21

21

21

21

21

21

21

Table 4: Litter and fetal weights - group mean values (g) on Day 29 of gestation

Group/Sex

 

Total litter weight

Male fetal weight

Female fetal weight

Overall fetal weight

Statistics test

 

Wi

Wi

Du

Wi

1F

Mean

349.7

40.0

38.2

39.3

 

SD

73.03

6.36

6.44

5.65

 

N

21

21

21

21

2F

Mean

324.1

41.8

41.9

41.9

 

SD

81.14

4.21

4.68

4.22

 

N

21

21

20

21

3F

Mean

320.5

40.2

39.9

39.8

 

SD

67.73

4.68

4.04

3.76

 

N

19

19

19

19

4F

Mean

286.6*

37.2

37.1

37.3

 

SD

81.94

6.78

6.67

6.07

 

N

21

21

21

21

Table 5: Fetal examinations

Group/Sex

 

Major abnormality findings (%)

Minor skeletal abnormalities and variations (%)

Minor visceral abnormalities/ variations and necropsy findings (%)

 

 

 

 

 

1F

N litters affected(%)

0 (0)

20 (95)

2 (9.5)

 

Total litters

21

21

21

 

N fetuses affected(%)

0 (0)

120 (63)

2 (1)

 

Total fetuses

190

190

190

 

 

 

 

 

2F

N litters affected(%)

3 (14)

21 (100)

3 (14)

 

Total litters

21

21

21

 

N fetuses affected(%)

3 (1.8)

109 (66)

4 (2.4)

 

Total fetuses

165

165

165

 

 

 

 

 

3F

N litters affected(%)

0 (0)

19 (100)

2 (10.5)

 

Total litters

19

19

19

 

N fetuses affected(%)

0 (0)

113 (73.3)

2 (1.2)

 

Total fetuses

154

154

154

 

 

 

 

 

4F

N litters affected(%)

3 (14)

21 (100)

4 (19)

 

Total litters

21

21

21

 

N fetuses affected(%)

4 (2.4)

134 (82.7)

4 (2.4)

 

Total fetuses

162

162

162

TAble 6: Historical control data (14 Studies, 2016-2017) - litter data

 

Corpora lutea

Implantations

Resorptions Early

Resorptions Late

Resorptions Total

Live young Male

Live young Female

Live young Total

Sex ratio (%M)

Pre-Implantation loss (%)

Post-Implantation loss (%)

Mean

9.8

8.7

0.6

0.3

0.9

4

3.8

7.8

50.9

12.9

10.7

Min

9

8

0.2

0.1

0.4

3.6

3.1

7

46.7

5.2

3.5

Max

10.5

10.2

1

0.9

1.8

4.8

5

9.8

57.4

20.2

18.9

Conclusions:
Oral administration of the test substance to New Zealand White rabbits from Day 6 to Day 28 of gestation at dose levels of 125, 250 or 500 mg/kg/day was associated with low maternal body weight gain and food consumption at 500 mg/kg/day.
Embryo-fetal survival, fetal and placental weight were unaffected by treatment and the fetal pathology findings at 500 mg/kg/day were minor skeletal variants which were not considered adverse.
It was therefore concluded that 250 mg/kg/day was the no observed effect level (NOEL) for both maternal toxicity and embryo-fetal development.
Executive summary:

The purpose of this study was to assess the influence of the test item on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of the New Zealand White rabbit.

Three groups of 22 females received the test item at doses of 125, 250 or 500 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle, purified water, at at the same volume dose as treated groups.

Animals were euthanized on Day 29 after mating for reproductive assessment and fetal examination. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

There were no signs were observed at either routine physical examination or in association with dose administration that could be related to treatment.

At 500 mg/kg/day the overall body weight gain and food consumption of females was low; approximately 17 % and 79% of Controls, respectively. Maternal body weight and food consumption at 125 or 250 mg/kg/day showed no adverse effects of treatment.

Macroscopic examination of females on GD29 did not reveal any findings that could be attributed to treatment with the test item. On Day 29 of gestation, mean numbers of implantations embryo-fetal survival, litter size and sex ratio were considered to be unaffected by treatment with the test item. Group mean placental weights, male or fetal weight and overall fetal weight were essentially similar to Controls at dose levels up to and including 500 mg/kg/day. The range and incidence of major abnormalities at 125 and 500 mg/kg/day showed no relationship to treatment. At 500 mg/kg/day there was a slight increased incidence of full supernumerary 13th ribs and unilateral caudal shift of pelvic girdle when compared with both the Concurrent Controls and the historical control data range. The combination of these skeletal variants at 500 mg/kg/day indicates a slight shift in rib/vertebral configuration which is not considered adverse.

Oral administration of the test item to New Zealand White rabbits from Day 6 to Day 28 of gestation at dose levels of 125, 250 or 500 mg/kg/day was associated with low maternal body weight gain and food consumption at 500 mg/kg/day. Embryo-fetal survival, fetal and placental weight were unaffected by treatment and the fetal pathology findings at 500 mg/kg/day were minor skeletal variants which were not considered adverse.

It was therefore concluded that 250 mg/kg/day was the no observed effect level (NOEL) for both maternal toxicity and embryo-fetal development.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

Prenatal development study in rabbit (key, K1):


The effect of the test substance on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of the New Zealand White rabbit were assessed. Three groups of 22 females received the test substance at doses of 125, 250 or 500 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating. A similarly constituted control group received the vehicle, purified water, at the same volume dose as treated groups. Animals were euthanized on Day 29 after mating for reproductive assessment and fetal examination. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination. 


The mean concentrations of test substance in test formulations analysed for the study were within +/-12% of nominal concentrations, confirming accurate formulation. There were no signs observed at either routine physical examination or in association with dose administration that could be related to treatment. 


At 500 mg/kg/day the overall body weight gain and food consumption of females was low; approximately 17% and 79% of controls, respectively. Maternal body weight and food consumption at 125 or 250 mg/kg/d showed no adverse effects of treatment. Macroscopic examination of females on GD29 did not reveal any findings that could be attributed to treatment with the test substance. On Day 29 of gestation, mean numbers of implantations embryo-fetal survival, litter size and sex ratio were considered to be unaffected by treatment with the test substance. Group mean placental weights, male or fetal weight and overall fetal weight were essentially similar to controls at dose levels up to and including 500 mg/kg/day. The range and incidence of major abnormalities at 125 and 500 mg/kg/day showed no relationship to treatment. At 500 mg/kg/day there was a slight increased incidence of full supernumerary 13th ribs and unilateral caudal shift of pelvic girdle when compared with both the concurrent controls and the historical control data range. The combination of these skeletal variants at 500 mg/kg/day indicates a slight shift in rib/vertebral configuration which is not considered adverse. 


In conclusion, oral administration of the test substance to New Zealand White rabbits from Day 6 to day 28 of gestation at dose levels of 125, 250 or 500 mg/kg/day was associated with low maternal body weight gain and food consumption at 500 mg/kg/day. Embryo-fetal survival, fetal and placental weight were unaffected by treatment and the fetal pathology findings at 500 mg/kg/day were minor skeletal variants which were not considered adverse. It was therefore concluded that 250 mg/kg/day was the NOEL for both maternal toxicity and embryo-fetal development.


A prenatal development study in rat will be considered after data interpretation and final reporting of the EOGRTS study in rat (ongoing).

Justification for classification or non-classification

Based on the available data and the criteria laid down in the CLP regulation, the test substance is considered not to be classified as reproductive toxicant.

Additional information