Fatty acids, C14-22, 2-ethylhexyl esters, epoxidized

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

An OECD 422 reproductive/developmental screening study using the 'target substance' identified no test item-related alterations in fertility or reproductive performance. No test item-related effects on natural delivery or litter endpoints were noted. The no-observed-adverse-effect level (NOAEL) for F0 and F1 males and females was provisionally established as 1000 mg/kg bw/d. (To be confirmed upon completion of anatomic pathology and thyroid hormone analyses).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2022 to present (study ongoing)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 2016:

Deviations:

yes

Remarks:

Inlife: Dose Admin. Food Consumption, Clinical Obs. & Phys. Examinations, Body Weights, Thyroxine Sample Collection, Clinical Path Sample Collection, Sample Handling. Postlife: Terminal BW's, Sacrifice, Macroscopic Obs. Tissue Collection & preservation.

Qualifier:

according to guideline

Guideline:

other: • United States Environmental Protection Agency-Office of Prevention, Pesticides & Toxic Substances (OPPTS) 870.3700

Version / remarks:

August 1998

Deviations:

yes

Remarks:

Inlife: Dose Admin. Food Consumption, Clinical Obs. & Phys. Examinations, Body Weights, Thyroxine Sample Collection, Clinical Path Sample Collection, Sample Handling. Postlife: Terminal BW's, Sacrifice, Macroscopic Obs. Tissue Collection & preservation.

Qualifier:

according to guideline

Guideline:

other: • United States Environmental Protection Agency 40 Code of Federal Regulations § 798.490 Developmental Toxicity

Deviations:

yes

Remarks:

Inlife: Dose Admin. Food Consumption, Clinical Obs. & Phys. Examinations, Body Weights, Thyroxine Sample Collection, Clinical Path Sample Collection, Sample Handling. Postlife: Terminal BW's, Sacrifice, Macroscopic Obs. Tissue Collection & preservation.

GLP compliance:

yes

Limit test:

no

Justification for study design:

Based on the ICH and in accordance with OECD 422, US EPA (OPPTS) 870.3700 and US EPA 40 Code of Federal Regulations § 798.490 Developmental Toxicity. This study is designed for use with the rat and is recommended by appropriate regulatory agencies.
Dose level selections were based upon the preliminary (oral) toxicity study in the Sprague-Dawley rat for 7 days (Labcorp Study Number 8484277; Labcorp Early Development Laboratories Inc., 2022). Male and female rats were administered 100, 300, or 1000 mg/kg/day for 7 days via oral gavage. All animals survived to their scheduled sacrifice, with no remarkable clinical observations noted. No remarkable effects on body weight or food consumption were observed. Increased seminal vesicle/coagulating gland weights and decreased ovary/oviduct weights were noted as the dose level increased; higher uterus/cervix weights were noted for animals administered 300 mg/kg/day. Macroscopic observations of discolored mandibular lymph nodes, large mandibular lymph nodes, small seminal vesicle, skin scab, and discolored thymus were noted in animals administered 1000 mg/kg/day; discolored thymus was also noted in one female administered 300 mg/kg/day. Based on this data, dose levels of 100, 300, and 1000 mg/kg/day, were selected which achieved 1000 mg/kg/day, the limit guideline dose. The oral route was chosen to simulate conditions of potential human exposure.

Group Dose Level (mg/kg/day) Dose Concentration (mg/mL) No. of Animals
Males
1 (Control) 0 0 12M + 12F
2 (Low) 100 10 12M + 12F
3 (Mid) 300 30 12M + 12F
4 (High) 1000 100 12M + 12F

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) of test material: Sponsor
- Lot/Batch No. OC061290 (also known as W061290)
- Purity, including information on contaminants, isomers, etc.: to be entered

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Exp. 31 March 2023
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: Formulations were stable for 8 days protected from light at 2-8°C. Stable in the vehicle control item at 10 and 750 mg/mL at room temperature, protected from light up to 24 hours and at 2 to 8 °C), and frozen (20℃ [±10 °C]) for up to 15 days under Labcorp Study 8484276

- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Formed a dosable suspension in corn oil.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): None

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: None

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

This study is designed for use with the rat and is recommended by appropriate regulatory agencies. The strain 'Sprague-Dawley' was selected based on historical control data and susceptibility to known developmental toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina, USA.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males) & 12 weeks (females)
- Weight at study initiation: 378 to 489g (males) and 202 to 324g (females).
- Fasting period before study: yes, F0 animals were fasted before sample collection.
- Housing: Males and females were group housed by sex (2-3/cage) in polycarbonate cages with Diamond Soft® bedding, except during 1: 1 male/female pairing. After mating had occurred (for females) or at the end of the pairing phase (for males and non-confirmed females), animals were individually housed in polycarbonate cages with bedding for the remainder of the study or until sacrifice. F0 females were housed with their litters during lactation.
- Diet (ad libitum): Certified Rodent Diet #2016C (Envigo RMS, Inc.), unless fasted for study procedures
- Water (ad libitum): yes
- Acclimation period: Males for 7 days prior to dose initiation and females for 7 days prior to predose estrous evaluation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 or more
- Photoperiod: 12-hour light/12-hour dark cycle.
IN-LIFE DATES: 26 January 2022 to 22 March 2022

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Prepared daily on each day of dosing, stored protected from light at refrigerated temperature (2 to 8°C). All dose formulations were prepared by Labcorp according to the mixing procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil, based on a previous 7-day study with the test item.
- Concentration in vehicle: Control group was administered corn oil only.
- Amount of vehicle (if gavage): Dosed at 10 mL/kg
- Batch No's: MKCM9808 (exp. 4 August 2023) & MKCP9878 (exp. 13 July 2024)

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2-week pairing phase
- Proof of pregnancy: vaginal copulatory plug or vaginal sperm referred to as day GD 0.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Housed individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from the middle of the vehicle control item (0 mg/mL) and each test item formulation (10, 30 & 100 mg/ml) prepared for administration on the first and last day of dosing.
All homogeneity and concentration verification results met acceptance criteria. The mean percent of target results were within 85 to 115%, and the relative standard deviation was not more than 10% and met specifications. No significant absorbance was detected in the control samples.

Duration of treatment / exposure:

At initiation of dosing, (P) males were 10 weeks old and (P) females were 12 weeks old. At initiation of pairing, (P) males were 12 weeks old. (P) Females were 10 weeks old at the initiation of predose estrous evaluations and 14 weeks old at pairing.

F0 males were dosed once daily via oral gavage for at least 14 days prior to pairing, throughout the 2-week pairing phase, and through the day prior to sacrifice for a minimum of 28 days. F0 females were dosed once daily for at least 14 days prior to pairing, throughout the pairing phase, and through gestation and lactation until Lactation Day (LD) 13. For females without confirmation of mating, dosing continued through 24 days after the last possible day of mating, when necessary.

Frequency of treatment:

Administration of the test item (in corn oil) by oral gavage administration for at least 4 weeks.

F0 males were dosed once daily via oral gavage for at least 14 days prior to pairing, throughout the 2-week pairing phase, and through the day prior to sacrifice for a minimum of 28 days. F0 females were dosed once daily for at least 14 days prior to pairing, throughout the pairing phase, and through gestation and lactation until Lactation Day (LD) 13. For females without confirmation of mating, dosing continued through 24 days after the last possible day of mating, when necessary.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control (Corn oil)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 males & 12 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose level selection was based upon the preliminary (oral) toxicity study in the Sprague-Dawley rat for 7 days (Labcorp Study Number 8484277; Labcorp Early Development Laboratories Inc., 2022). Male and female rats were administered 100, 300, or 1000 mg/kg/day for 7 days via oral gavage. All animals survived to their scheduled sacrifice, with no remarkable clinical observations noted. No remarkable effects on body weight or food consumption were observed. Increased seminal vesicle/coagulating gland weights and decreased ovary/oviduct weights were noted as the dose level increased; higher uterus/cervix weights were noted for animals administered 300 mg/kg/day. Macroscopic observations of discolored mandibular lymph nodes, large mandibular lymph nodes, small seminal vesicle, skin scab, and discolored thymus were noted in animals administered 1000 mg/kg/day; discolored thymus was also noted in one female administered 300 mg/kg/day. Based on this data, dose levels of 100, 300, and 1000 mg/kg/day were selected which achieved 1000 mg/kg/day, the limit guideline dose. The oral route was chosen to simulate conditions of potential human exposure.
- Fasting period before blood sampling for clinical biochemistry: F0 animals fasted for sample collection.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Performed for each animal during the dosing interval at ca. 0.5, 1, and 4 hours postdose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed twice daily (a.m. and p.m.) for mortality, abnormalities, and signs of pain or distress.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed once prior to dose initiation at randomization, predose on the first day of dosing, and once weekly thereafter, including the day of sacrifice.
Females were weighed at least once prior to dose initiation at randomization, predose on the first day of dosing, and once weekly until confirmation of mating, as applicable. In addition, body weights were also recorded on GD 0, 7, 14, and 20 (confirmed mated females) and on LD 1, 4, 7, 11, and 13 (day of sacrifice). For the females without confirmation of mating, body weight measurements continued weekly until sacrifice (in the raw data but not reported).

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated using daily vaginal lavage for 2 weeks prior to dose initiation. Estrous cycle stages continued to be recorded for the first 2 weeks of dosing, prior to pairing (total of 15 samples in the pre mating phase). Estrous cycle evaluations continued during pairing until the day positive signs of mating were observed for each female. Estrous cycle determination was also performed on the day of necropsy for scheduled and unscheduled sacrifices. Estrous slides were read fresh and were not stained or retained.

Sperm parameters (parental animals):

Parameters examined in F0 males: testis weight, epididymis weight, (SVC) Seminal Vesicle/Coagulating gland & prostate

Testes: Detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify test item-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells, or sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

Daily records of mortalities and consequent changes in litter sizes were maintained. Observations were recorded by exception. When possible, pups found dead or in a moribund condition were taken to necropsy. Pups found cannibalized were taken to necropsy.

Body weights and detailed observations were recorded on PND 4, 7, 11, and 13.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
All F1 animals (except for pups selected for T4 analysis without abnormal clinical observations) had an examination of the external features of the carcass; external body orifices; cervical, cranial, abdominal, and thoracic viscera; organs; and tissues conducted to the extent possible. Any macroscopic abnormalities were noted.

The following parameters were examined in F1 offspring:

Postnatal Day (PND) 0 Evaluations:
When delivery was complete, the number of live, dead, cannibalized, and stillborn pups was recorded. Dead and cannibalized pups were taken to necropsy.

PND 1 Evaluations: On PND 1, the number of live, dead, and cannibalized pups was recorded. Sex, body weight, and individual observations (abnormal only) were recorded for live pups. Dead and cannibalized pups were taken to necropsy.

Litter Observations: Daily records of mortalities and consequent changes in litter sizes were maintained. Observations were recorded by exception. When possible, pups found dead or in a moribund condition were taken to necropsy. Pups found cannibalized were taken to necropsy.

Ano-Genital Distance Measurement: On PND 1, each male and female pup was measured for ano-genital distance and a value in (mm) to two decimal places was recorded.

Nipple Count: On PND 13, the number of nipples observed for each male pup was recorded.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: F0 males were dosed once daily via oral gavage for at least 14 days prior to pairing, throughout the 2-week pairing phase, and through the day prior to sacrifice for a minimum of 28 days. All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: F0 females were dosed once daily for at least 14 days prior to pairing, throughout the pairing phase, and through gestation and lactation until Lactation Day (LD) 13 (see Protocol Deviations). For females without confirmation of mating, dosing continued through 24 days after the last possible day of mating, when necessary.

GROSS NECROPSY:
All F0 and F1 animals (except pups selected for T4 analysis without abnormal clinical observations) had an examination of the external features of the carcass; external body orifices; cervical, cranial, abdominal, and thoracic viscera; organs; and tissues conducted to the extent possible. Any macroscopic abnormalities were noted.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination and weighed, respectively.
Adrenals(2), Brain (including cerebrum, cerebellum, and pons), Cervix, Coagulating gland, Epididymides(2), Heart (including auricular and ventricular regions), Kidney(2), Liver, Ovary(2), Oviduct(2), Pituitary, Prostate, Seminal vesicles(2), Spleen, Testes(2), Thymus, Thyroid (2 lobes), with parathyroid, Uterus

Postmortem examinations (offspring):

SACRIFICE
F1 Pups Unscheduled Sacrifices: Pups found dead on PND 0 had the lungs floated to determine whether death occurred before (stillborn) or after birth. Tissues that could not be examined were noted.

F1 Pups Scheduled Sacrifices: For pups selected for thyroid hormone analysis, sacrifice was on PND 4; the carcass was discarded without necropsy following blood collection for thyroid analysis, as applicable. Surviving pups were sacrificed on PND 13

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Any macroscopic abnormalities were noted.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated were prepared for microscopic examination and weighed, respectively.
Thyroid (2 lobes) with parathyroid

Statistics:

The pairwise comparisons of interest were: Group 1 vs Groups 2, 3, and 4.
Means and standard deviations were calculated. All statistical tests were evaluated at the 5.0, 1.0, and 0.1% probability levels.
Analysis of variance (ANOVA) and pairwise comparisons, as applicable, were used to analyze the following.
• Body weight (F0 animals)
• Body weight change (F0 animals)
• Food consumption (F0 animals)

Ano-genital distance was analyzed using analysis of covariance (ANCOVA). Cube root litter weight was taken as the covariate within the analysis.

The following data were statistically analyzed.
• Mean number of estrous cycles and mean cycle length - Procedure III - A Kruskal-Wallis nonparametric ANOVA.
• The percentage of females with stillbirths – Procedure IV (one-sided upper tail).
• Mean live pup weights (male and female); litter size (live and dead pups) was used as the covariate – Procedure II - Analysis of covariance (ANCOVA)
• Pup survival indices – Procedure III - A Kruskal-Wallis nonparametric ANOVA.
• Male and female mating, fecundity, and fertility indices, and parturition indices – Procedure IV (one sided lower tail)
Absolute organ weights, organ:terminal body weight ratios, and organ:brain weight ratios – Procedure I (ANOVA) - Levene’s test was conducted to test for equality of variances between groups.
Note: Females on LD 14 and males on Post-Pairing Day 9s, scheduled sacrifices only, are included in the organ weight summary table with statistical analysis.
• Continuous clinical pathology data – Procedure I (ANOVA) - Levene’s test was conducted to test for equality of variances between groups.
• Thyroid hormone analysis (pups of both sexes), where a single sample/pup was obtained (no analysis was performed for pooled samples) – Procedure I (ANOVA) - Levene’s test was conducted to test for equality of variances between groups.
• Continuous FOB data – Procedure I (ANOVA) - Levene’s test was conducted to test for equality of variances between groups.

Reproductive indices:

Individual Breeding Pairs: Pregnant, Not Confirmed/Pregnant, Not Pregnant, Not Confirmed/Not Pregnant

Male Reproductive performance: Mating Index (%), Fecundity Index (%), Fertility Index (%)
Mating index % = (Number of males mating with at least 1 female / Number of males cohabitated with at least 1 female) x 100
Fecundity index % = (Number of males impregnating at least 1 female / Number of males mating with at least 1 female) x 100
Fertility Index % = (Number of males impregnating at least 1 female / Number of males cohabitated with at least 1 female) x 100

Female Reproductive Performance: Mating Index %, Fecundity Index, %Fertility Index %
Mating index % = Mated females/females cohabited (excluding females sacrificed during Cohabitation) x 100
Fecundity Index % = Pregnant females/mated females (excluding females with an undetermined pregnancy status) x 100
Fertility Index % = Pregnant females/females cohabited (excluding females sacrificed during Cohabitation or with an undetermined pregnancy status) x 100

Offspring viability indices:

For pup survival indices the following were calculated:
Livebirth Index
Day 4 Viability Index

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three F0 control females (R0402, R0405, and R0412) were sacrificed early in a moribund condition. Animal R0402 (on GD 22) and R0405 (on GD 23) due to severe clinical observations of decreased general activity, liquid feces, piloerection, body temperature cool to touch, abnormal eye color, and/or swollen vulva area. Animal R0412 (on LD 3) due to severe clinical observations of liquid feces, walking on toes, piloerection, hunched posture, and not attending to litters; as a result, the litter was also sacrificed.
One F0 female at 100 mg/kg/day (R0501) was sacrificed on LD 0 due to total litter loss. One F0 female at 300 mg/kg/day (R0609) was sacrificed on GD 23 due to gavage error.
One F0 female at 300 mg/kg/day (R0610) was sacrificed on LD 1 due to severe clinical observations of decreased general activity, ataxia, piloerection, thin physical appearance, hunched posture, labored respiration, and body temperature cool to touch. This animal did not attend the litter; thus, the litter was sacrificed on the same day. Two F0 females at 1000 mg/kg/day (R0705 and R0711) were sacrificed early at an unscheduled interval on LD 4 or 0, respectively, due to severe clinical observations of decreased general activity, piloerection, thin physical appearance, hunched posture, body temperature cool to touch, or liquid feces. The litters of these two F0 females were sacrificed on the same day as the dams. All animals sacrificed early at an unscheduled interval had similar clinical observations, including controls, and the number of animals sacrificed at an unscheduled interval were similar among groups; therefore, they were considered not test item related. All other F0 females survived until their scheduled sacrifice.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

F0 thyroid hormone analysis pending

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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

Remarks:

Provisional, awaiting final thyroid analysis results.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

F1 pups that died or were sacrificed at an unscheduled interval were so because their respective F0 females died or were sacrificed at an unscheduled interval.
All other toxicity F1 pups survived to their scheduled sacrifice.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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:

no effects observed

Description (incidence and severity):

Pup thyroid hormone analysis pending

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weight differences noted in F1 pups sacrificed on PND 13 were decreased thyroid/parathyroid weights in female pups from the group administered 1000 mg/kg/day. This finding was not noted in males nor were microscopic correlates noted, thus, considered non-adverse.
All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body or brain weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Remarks:

Provisional, awaiting final thyroid analysis results.

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Results of Homogeneity and Concentration Verification Analyses

Interval	Location	Replicate	Lankroflex ED6 in Corn Oil
			0 mg/mL		10 mg/mL		30 mg/mL		100 mg/mL
			Actual	% of Target	Actual	% of Target	Actual	% of Target	Actual	% of Target
First day	Top	1	-	-	8.87	88.73	-	-	89.5	89.5
TA 1		2	DNS	-	9.19	91.95	-	-	90.7	90.7
	Middle	1	-	-	9.24	92.40	27.3	91.2	89.1	89.1
		2	-	-	9.17	91.70	26.9	89.5	90.1	90.1
		3	-	-	-	-	27.2	90.7	-	-
	Bottom	1	-	-	8.81	88.06	-	-	90.7	90.7
		2	-	-	9.06	90.62	-	-	88.9	88.9
		Mean	-	-	-	90.58	-	90.4	-	89.9
		RSD (%)	-	-	-	2.0	-	0.9	-	0.9
	Top	1	-	-	9.23	92.27	-	-	88.3	88.3
TA 38		2	DNS	-	9.28	92.76	-	-	89.9	89.9
	Middle	1	-	-	9.04	90.35	-	-	88.9	88.9
		2	-	-	8.95	89.47	-	-	89.9	89.9
		3	-	-	-	-	-	-	-	-
	Bottom	1	-	-	9.04	90.43	-	-	88.7	88.7
		2	-	-	9.39	93.86	-	-	90.4	90.4
		Mean	-	-	-	91.53	-	-	-	89.4
		RSD (%)	-	-	-	1.9	-	-	-	0.9
	Top	1	-	-	9.09	90.95	-	-	-	-
TA 54		2	DNS	-	9.24	92.45	-	-	-	-
	Middle	1	-	-	9.24	92.41	27.0	90.1	91.1	91.1
		2	-	-	9.41	94.15	27.9	92.9	92.1	92.1
		3	-	-	-	-	27.5	91.7	91.3	91.3
	Bottom	1	-	-	9.38	93.81	-	-	-	-
		2	-	-	9.42	94.22	-	-	-	-
		Mean	-	-	-	93.00	-	91.6	-	91.5
		RSD (%)	-	-	-	1.4	-	1.6	-	0.6
- = Not applicable DNS = Detection not significant RSD = Relative standard deviation

Summary of Clinical Observations

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg	(0)	100	300	1000
Phase: Gestation
Category Observation	Group / Sex	1/F	2/F	3/F	4/F
	No. in Group	12	11	12	11
Activity
general activity, decreased		2	0	1	0
Discharge
source known, vulva, red		0	0	1	0
Excretion
liquid feces, individual observation		2	0	0	0
Eyes
abnormal color, eyes, both, pale		1	0	0	0
Pelage
hair loss, feet, front both		0	0	0	1
piloerection		1	0	0	0
rough		1	0	0	0
thinning, leg, hind left		0	0	0	1
thinning, ventral thoracic, left		0	0	0	1
Physical Appearance
swollen area, vulva		1	0	1	0
Removal
removal approved by, veterinarian		2	0	1	0
Temperature
cool to the touch, whole body		2	0	1	0
Unscheduled Study Removal
severity of clinical observations		2	0	1	0

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg	(0)	100	300	1000
Phase: Lactation
Category Observation	Group / Sex	1/F	2/F	3/F	4/F
	No. in Group	9	10	11	9
Activity
general activity, decreased		0	0	1	1
Behavior   other
dam not attending to litter		1	0	0	1
Discharge
source known, vulva, red		0	1	0	0
unable to produce milk		0	0	1	1
Excretion
liquid feces, individual observation		1	0	0	1
Gait
ataxia		0	0	1	0
walking on toes		1	0	0	0

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg	(0)	100	300	1000
Phase: Lactation
Category Observation	Group / Sex	1/F	2/F	3/F	4/F
	No. in Group	9	10	11	9
Pelage
abnormal color, uro genital area, brown		0	0	1	1
hair loss, abdomen, left		0	0	0	1
hair loss, feet, front both		0	0	0	1
hair loss, leg, hind left		0	0	0	1
hair loss, legs, front both		0	0	0	1
piloerection		1	0	1	1
rough		0	1	0	0
thinning, abdomen, left		0	0	0	1
thinning, leg, hind right		0	0	0	1
thinning, uro genital area		0	0	0	1
Physical Appearance
thin		0	0	1	1
Posture
hunched		1	0	1	1
Removal
removal approved by, SD		0	1	0	0
removal approved by, vet		1	0	1	2
Respiration
labored		0	0	1	0
Temperature
cool to the touch, whole body		0	1	1	1
Unscheduled Study Removal
severity of clinical observations		1	0	1	2
total litter loss		0	1	0	0

Summary of Body Weight

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg	(0)	100	300	1000
Data presented in ‘g’
Phase		PM			PR	PP
Group / Sex		Day 0	7	14	7	0	7	9
1/M	Mean	427	455	493	511	542	556	570
	SD	23.6	26.8	34.2	34.2	36.9	40.1	43.6
	N	12	12	12	12	12	12	12
2/M	Mean	427	461	499	520	557	575	588
	SD	24.1	28.8	33.8	37.5	44.6	48.6	52.1
	N	12	12	12	12	12	12	12
3/M	Mean	426	462	498	516	549	563	575
	SD	24.7	27.6	32.1	37.2	40.2	41.2	42.7
	N	12	12	12	12	12	12	12
4/M	Mean	425	460	498	514	543	555	566
	SD	30.4	31.6	38.3	39.6	43.2	46.0	48.9
	N	12	12	12	12	12	12	12
	Statistics	A	A	A	A	A	A	A

PM = Premating
PR = Pairing
PP = Post Pairing
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg	(0)	100	300	1000
Data presented in ‘g’
Phase		PM
Group / Sex		Day 0	7	14
1/F	Mean	252	258	267
	SD	34.4	34.8	34.4
	N	12	12	12
2/F	Mean	254	262	270
	SD	32.9	34.1	38.3
	N	12	12	12
3/F	Mean	253	260	271
	SD	33.3	34.3	36.4
	N	12	12	12
4/F	Mean	248	254	261
	SD	23.8	24.1	27.3
	N	12	12	12
	Statistics	A	A	A

PM = Premating
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg	(0)	100	300	1000
Data presented in ‘g’
Phase		GE				LA
Group / Sex		Day 0	7	14	20	1	4
1/F	Mean	279	307	342	404	303	299
	SD	36.3	41.5	44.6	43.1	40.8	42.2
	N	10	10	10	10	8	8
2/F	Mean	269	300	329	402	300	296
	SD	30.9	36.7	40.3	51.2	51.2	51.0
	N	10	10	10	10	9	9
3/F	Mean	284	315	351	422	311	310
	SD	36.9	40.7	42.0	47.2	43.6	35.5
	N	9	9	9	9	10	9
4/F	Mean	271	298	331	395	290	286
	SD	29.4	27.9	29.6	34.0	19.5	15.9
	N	9	9	9	9	8	8
	Statistics	A	A	A	A	A	A

GE = Gestation
LA = Lactation
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg	(0)	100	300	1000
Data presented in ‘g’
Phase		LA
Group / Sex		Day 7	13
1/F	Mean	304	319
	SD	33.3	31.2
	N	8	8
2/F	Mean	298	308
	SD	44.4	41.9
	N	9	9
3/F	Mean	310	319
	SD	35.5	23.6
	N	9	9
4/F	Mean	291	300
	SD	15.9	23.3
	N	7	7
	Statistics	A	A

LA = Lactation
A = ANOVA and Dunnett's

 

Summary of Food Consumption

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg	(0)	100	300	1000
Data presented in ‘g’
Phase		PM
Group / Sex		Day 0-3	3-7	7-10	10-14	0-14
1/F	Mean	38	48	34	48	168
	SD	4.4	6.6	4.2	5.1	18.3
	N	6	6	6	6	6
2/F	Mean	40	52	35	46	172
	SD	4.5	5.3	4.4	4.4	15.9
	N	6	6	6	6	6
3/F	Mean	38	48	34	46	166
	SD	4.8	4.5	4.5	3.7	16.7
	N	6	6	6	6	6
4/F	Mean	36	49	35	49	168
	SD	4.2	7.2	4.4	5.0	19.4
	N	6	6	6	6	6
	Statistics	A	A	A	A	A

PM = Premating
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg	(0)	100	300	1000
Data presented in ‘g’
Phase		GE
Group / Sex		Day 0-7	7-14	14-20	0-20
1/F	Mean	106	124	116	348
	SD	15.0	18.8	15.1	43.5
	N	10	10	9	9
2/F	Mean	109	115	112	335
	SD	17.9	23.8	25.3	63.8
	N	11	11	11	11
3/F	Mean	110	126	111	348
	SD	12.7	20.1	21.2	49.5
	N	9	9	9	9
4/F	Mean	115	117	104	336
	SD	14.6	21.4	15.4	44.4
	N	11	11	11	11
	Statistics	A	A	A	A

GE = Gestation
A = ANOVA and Dunnett's

 

Test Item	(dosage)	1	2	3	4
Lankroflex ED6	mg/kg	(0)	100	300	1000
Data presented in ‘g’
Phase		LA
Group / Sex		Day 1-4	4-7	7-11	11-13	1-13
1/F	Mean	67	109	172	106	454
	SD	13.2	24.6	25.2	19.8	70.1
	N	8	8	8	8	8
2/F	Mean	56	96	151	91	394
	SD	25.4	28.3	38.6	25.5	111.6
	N	9	9	9	9	9
3/F	Mean	63	97	154	95	410
	SD	36.9	46.1	51.2	24.2	147.6
	N	9	9	9	9	9
4/F	Mean	52	99	148	92	398
	SD	25.0	15.9	36.9	22.6	87.1
	N	8	7	7	7	7
	Statistics	A	A	A	A	A

LA = Lactation
A = ANOVA and Dunnett's

 

Summary of Fertility and Reproductive Performance

Summary of Male Reproductive Performance

Treatment group	Control	100 mg/kg	300 mg/kg	1000 mg/kg
Total Males	12	12	12	12
Unscheduled Deaths Prior to Cohabitation	0	0	0	0
Males Cohabitated	12	12	12	12
Unscheduled Deaths During Cohabitation	0	0	0	0
Males mating with at least 1 female	12	11	12	11
Males impregnating at least 1 female	12	10	11	9
Mating Index (%)	100	92	100	92
Fecundity Index (%)	100	91	92	82
Fertility Index (%)	100	83	92	75

Mating index %  =  (No. of males mating with at least 1 female / No. of males cohabitated with at least 1 female) x 100
Fecundity index %  =  (No. of males impregnating at least 1 female / No. of males mating with at least 1 female) x 100
Fertility Index %  =  (No. of males impregnating at least 1 female / No. of males cohabitated with at least 1 female) x 100

 

Summary of Female Reproductive Performance

Treatment group	Control	100 mg/kg	300 mg/kg	1000 mg/kg
Total Females	12	12	12	12
Unscheduled Deaths Prior to Cohabitation	0	0	0	0
Females Cohabitated	12	12	12	12
Unscheduled Deaths During Cohabitation	0	0	0	0
Females Mated	12	11	12	11
Pregnant Females	12	10	11	9
Accidental	0	0	1	0
Moribund Sacrifice	3	0	1	2
Pup(s) / Total Litter Dead	0	1	0	0
Non Pregnant Females	0	2	1	3
Matings Per Day Periods Of Cohabitation
Day 1	2	4	3	1
Day 2	2	1	0	2
Day 3	3	4	6	2
Day 4	2	2	0	4
Day 5	1	0	1	0
Day 14	0	0	0	2
Mating Index %	100	92	100	92
Fecundity Index %	100	91	92	82
Fertility Index %	100	83	92	75

Mating index  % = Mated females/females cohabited (excluding females sacrificed during Cohabitation) x 100
Fecundity Index  % = Pregnant females/mated females (excl. females with an undetermined pregnancy status) x 100
Fertility Index % = Pregnant females/females cohabited (excl. females sacrificed during Cohabitation or with an undetermined pregnancy status) x 100

 

Summary of Natural Delivery and Litter Data

	Dose level	Control (0) mg/kg/day		100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
Females:  Mated	N	12		11	12	10
Pregnant	N	12		10	11	9
	%	100		91	92	90
Delivering	N	9		10	10	9
	%	75		100	91	100
Duration of Gestation ^a:	MEAN	22.9		23.1	23.4	23.4
	S.D.	0.7		0.3	0.5	0.5
	N	7		10	8	9
Females with Liveborn pups	N	9		9	10	9
Gestation Index	%	75		90	91	100
with Stillborn pups	N	1		1	0	1
	%	11.11		11.11	0.00	11.11
females with no liveborn pups	N	0		1	0	0
	%	0.00		10.00	0.00	0.00
pups Delivered		111		134	141	114
	MEAN	12.33		13.40	14.10	12.67
	S.D.	2.29		2.17	3.00	3.24
	N	9		10	10	9
Liveborn		110		122	141	113
	MEAN	12.22		12.20	14.10	12.56
	S.D.	2.54		4.83	3.00	3.28
Stillborn		1		12	0	1
	MEAN	0.11		1.20	0.00	0.11
	S.D.	0.33		3.46	0.00	0.33
Uncertain		0		0	0	0
Implantation Sites ^b		136		127	174	125
	MEAN	12.36		14.11	15.82	13.89
	S.D.	3.56		1.90	1.72	3.41
	N	11		9	11	9
PostImplantation Loss	MEAN	0.75		0.56	1.80	1.33
	S.D.	0.89		0.73	2.70	1.87
	%	6.11		4.38	11.13	8.89

a= Only animals that were confirmed mated and produced a litter are included in the analysis of "Duration of Gestation"
b = Animals R0412 and R0510 were excluded from analysis of “Implantation Sites”.
N = Number of Females or LittersTOTAL = Number of pups or Implants

 

	Dose level	Control (0) mg/kg/day		100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
Females:  Delivering Live pups		9		9	10	9
Died or Killed During Lactation		1		0	1	2
Removed due to Total Litter Loss		0		1	0	0
Surviving to Scheduled Sacrifice		8		9	9	7
pup Survival Indices
Livebirth Index	MEAN%	99		89	100	99
Day 4 Viability Index	MEAN%	86		75	84	80
pup Disposition
Culled day 4	TOTAL	0		0	0	0
Dead Pup		2		2	4	24
Moribund Pup Sacrifice		10		1	13	0
Pup Removed/Dam Unscheduled Death		0		0	0	6
Other		0		0	1	0
Pup Sacrifice and Discard		13		17	16	10
Cannibalized		0		0	0	1
Missing		2		5	4	6
pups Surviving at 13 days	TOTAL	83		97	103	66
pups Dead Pup, Moribund Pup Sacrifice, Other, Pup Sacrifice and Discard, Missing and/or Cannibalized+
Days 0 4		27		20	36	41
Days 5 -13		0		5	2	0
Entire Litter Dead Pup, Moribund Pup Sacrifice, Other, Pup Sacrifice and Discard, Missing and/or Cannibalized+
Days 0 4	N	1		0	1	1
Days 5 -13	N	0		0	0	0

N = Number of Females or LittersTOTAL = Number of pups or Implants + Excludes pups where the dam has died or was killed moribund

 

Summary of Pup Survival

Dose level		Control (0) mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
Total Number and Mean Males Percent by Litter
Day 1		50	58	56	43
	MEAN%	45	48	41	43
Day 13		37	45	46	28
	MEAN%	44	44	45	39
Live pups/Litter with Live pups
Day 1	MEAN	12.11	13.44	13.10	11.38
	S.D.	2.47	2.35	3.57	3.85
	N	9	9	10	8
Day 4	MEAN	11.13	12.22	12.67	8.88
	S.D.	2.30	2.39	2.55	4.16
	N	8	9	9	8
Day 7	MEAN	10.38	11.33	11.44	9.43
	S.D.	1.85	2.18	2.24	2.44
	N	8	9	9	7
Day 11	MEAN	10.38	10.78	11.44	9.43
	S.D.	1.85	3.03	2.24	2.44
	N	8	9	9	7
Day 13	MEAN	10.38	10.78	11.44	9.43
	S.D.	1.85	3.03	2.24	2.44
	N	8	9	9	7

N = Number of Litters.

 

Summary of Pup Clinical Observations

Dose Gp: Dose level: Number of Litters Evaluated: Number of pups Evaluated:		Control (0) mg/kg/day 9 108		2 100 mg/kg/day 9 119		3 300 mg/kg/day 10 135		4 1000 mg/kg/day 9 84
Category, Observation		a	b	a	b	a	b	a	b
Pelage, thinning
	pups	0	0	0	0	0	0	18	18
	Litters	0		0		0		2
Physical Appearance, missing, tail, distal
	pups	0	0	0	0	1	7	0	0
	Litters	0		0		1		0
Physical Appearance, missing milk band
	pups	12	22	0	0	13	13	3	6
	Litters	1		0		1		1
Physical Appearance, thin
	pups	0	0	13	56	23	139	4	7
	Litters	0		2		2		2

Note: a = Number of animals with Observation
b = Number of days Observation seen

 

Dose Gp: Dose level: Number of Litters Evaluated: Number of pups Evaluated:		Control (0) mg/kg/day 9 108		2 100 mg/kg/day 9 119		3 300 mg/kg/day 10 135		4 1000 mg/kg/day 9 84
Category, Observation		a	b	a	b	a	b	a	b
Removal, removal approved by
	pups	10	10	1	1	14	14	7	7
	Litters	1		1		2		1
Respiration, labored
	pups	0	0	1	1	0	0	0	0
	Litters	0		1		0		0
Skin, abnormal color
	pups	0	0	3	6	0	0	8	8
	Litters	0		1		0		2
Skin, scab
	pups	0	0	1	1	1	7	0	0
	Litters	0		1		1		0

Note: a = Number of animals with Observation
b = Number of days Observation seen

 

Dose Gp: Dose level: Number of Litters Evaluated: Number of pups Evaluated:		Control (0) mg/kg/day 9 108		2 100 mg/kg/day 9 119		3 300 mg/kg/day 10 135		4 1000 mg/kg/day 9 84
Category, Observation		a	b	a	b	a	b	a	b
Temperature, cool to the touch
	pups	12	12	1	1	13	13	7	7
	Litters	1		1		1		1
Unscheduled Study Removal, accidental trauma
	pups	0	0	0	0	1	1	0	0
	Litters	0		0		1		0
Unscheduled Study Removal, sacrificed following dosing procedure
	pups	0	0	0	0	1	1	0	0
	Litters	0		0		1		0
Unscheduled Study Removal, severity of clinical observations
	pups	10	10	1	1	12	12	7	7
	Litters	1		1		1		1

Note: a = Number of animals with Observation
b = Number of days Observation seen

 

 

 

 

 

 

Conclusions:

Adult F0 female and male rats were administered the vehicle control (corn oil) or 100, 300, or 1000 mg/kg/day Lankroflex ED6 by oral gavage for at least 4 weeks. No test item related mortality; clinical observations; alterations in mean body weight or mean food consumption; or effects on neurobehavioral evaluations, reproductive performance, natural delivery or litter endpoints, or clinical pathology endpoints were noted. Test item related, but non-adverse (pending further confirmation when data are available), lower mean pup body weight was noted in a dose-dependent manner. No other test item related pup evaluation endpoints, including clinical observations, ano-genital distance, or nipple/areolae counts, were noted. No observed adverse effect levels (NOAELs) will be determined upon completion of anatomic pathology and thyroid hormone analyses.

Executive summary:

This study assessed the general systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints in line with OECD 422 following oral (gavage) administration of Lankroflex ED6 (Fatty acids, C14-22, 2‑ethylhexyl esters, epoxidized) for at least 4 weeks. Adult F0 female and male rats were administered the vehicle control (corn oil) or the test item at 100, 300, or 1000 mg/kg/day.

Assessment of toxicity was based on mortality, clinical observations, body weights, food consumption, neurobehavioral evaluations, reproductive performance, and necropsy findings. Blood samples were collected for hormone and clinical pathology evaluations.

No test item-related mortality was noted. No test item-related postdose observations or clinical observations were noted. No test item-related alterations in mean body weight or mean food consumption were noted. No test item-related effects on neurobehavioral evaluations, including detailed clinical observations, quantitative assessments, functional observational battery (FOB) assessments, or motor activity were noted. No test item-related alterations in estrous cycle were noted. No test item-related alterations in fertility or reproductive performance were noted. No test item-related effects on natural delivery or litter endpoints were noted.

Test item-related, but non-adverse (pending further confirmation when thyroid data are available), lower mean pup body weight was noted for pups from F0 animals administered 100, 300, or 1000 mg/kg/day, compared with controls. No test item-related pup clinical observations or alterations in ano-genital distance were noted. No nipple/areolae counts were noted for any male pups examined.

No test item-related clinical pathology effects were observed. No test item-related macroscopic observations were noted for F0 or F1 animals.

Test item-related, but non-adverse, organ weight effects were noted for the liver and thyroid of males administered ≥300 mg/kg/day and the thyroid, thymus, and/or uterus/cervix of females administered ≥100 mg/kg/day. Test item-related, but non-adverse, microscopic microscopic findings in animals administered 1000 mg/kg/day were noted in the kidney, liver, and thyroid of males and the thyroid of females.

Test item-related, but non-adverse, decreased thyroid/parathyroid weight was noted in F1 PND 13 female pups from females administered 1000 mg/kg/day. This finding was not noted in males, nor were microscopic correlates noted.

In conclusion, adult F0 female and male rats administered the vehicle control (corn oil) or 100, 300, or 1000 mg/kg/day Lankroflex ED6 by oral gavage for at least 4 weeks. No test item-related mortality; clinical observations; alterations in mean body weight or mean food consumption; or effects on neurobehavioral evaluations, reproductive performance, natural delivery or litter endpoints, or clinical pathology endpoints were noted. Test item‑related, but non-adverse (pending further confirmation when data are available), lower mean pup body weight was noted in a dose-dependent manner. No other test item‑related pup evaluation endpoints, including clinical observations, ano-genital distance, or nipple/areolae counts, were noted. Lankroflex ED6-related, but non-adverse organ weight effects were noted for the liver and thyroid of males administered ≥300 mg/kg/day and the thyroid, thymus, and/or uterus/cervix of females administered ≥100 mg/kg/day. Lankroflex ED6-related, but non-adverse microscopic findings in animals administered 1000 mg/kg/day were noted in the kidney, liver, and thyroid of males and the thyroid of females. Lankroflex ED6-related, but non-adverse decreased thyroid/parathyroid weight was noted in F1 female pups from females administered 1000 mg/kg/day. Based on these findings, a provisional no-observed-adverse-effect level (NOAEL) for F0 and F1 males and females was 1000 mg/kg/day. NOAEL will be determined upon completion of thyroid hormone analysis.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1 (reliable without restriction)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The 'target' substance did not induce any adverse toxic effects in parent males or females and did not identify any test item-related alterations in fertility, reproductive performance or impairment of the development of the F1 offspring. The no-observed-adverse-effect level (NOAEL) for F0 and F1 males and females was established as 1000 mg/kg bw/d. (To be confirmed upon completion of anatomic pathology and thyroid hormone analyses).

 

 

 

Effects on developmental toxicity

Description of key information

In an OECD 414 study in pregnant rabbits administered the 'target substance' at up to 750 mg/kg/day during the period of gestation (GD 6 through 28) a no observed adverse effect level (NOAEL) was established at 750 mg/kg/day, the highest dose tested.

In an OECD 414 study in pregnant rats (second species) oral administration of a read-across 'source substance' to rats from implantation to one day prior to the expected day of parturition (GD 6-19) at up to 1000 mg/kg bw/d, the limit dose, caused neither evidence of maternal nor developmental toxicity. The no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity was established as 1000 mg/kg bw/d.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015/2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Batch identification: CE80580015
Content: 99.9% (100% - water content)
Storage stability: Expiry date: 01 May 2016
Physical state/appearance: Liquid/ colorless, clear

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation:
- Housing: single caging
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 1st cohort From: 2015-07-09 To: 2015-07-29, 2nd cohort: from 2015-07-10 to 2015-07-30

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The oily test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed with a magnetic stirrer until it is dissolved. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): test item is insoluble in water.
- Concentration in vehicle: adjusted to amount of vehicle
- Amount of vehicle (if gavage): 4mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in corn oil at room temperature over a period of 7 days had been verified prior to the start of the study. Given that test substance is completely miscible with corn oil Ph. Eur./DAB, solutions are considered to be homogenous without further analysis.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: referred to as day 0 of pregnancy

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Duration of test:

GD 6-19 / 14 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: outcome of 28d and 90d repeated dose studies (no adverse findings, NOAEL 1000 mg/kg bw)

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Time schedule: Mortality/Morbidity, pertinent behavioral changes and/or signs of overt toxicity. were checked twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GO 0 to 20).

DETAILED CLINICAL OBSERVATIONS:
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-20).

FOOD CONSUMPTION:
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

BODY WEIGHT:
Time schedule for examinations: GO 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day: GD 20.
- On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order.
- After the dams had been sacrificed, they were necropsied and assessed for gross pathology, in randomized order. The uteri and the ovaries were removed and the following data were recorded: weight of the unopened uterus, number of corpora lutea, number and distribution of implantation sites classified as live fetuses and dead implantations.
Dead implantations include:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

- After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.

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: Site of implantations in the uterus

Fetal examinations:

- External examinations: all per litter
- Soft tissue examinations: half per litter
- Skeletal examinations: half per litter

EXAMINATIONS OF THE FETUSES AFTER DISSECTION FROM THE UTERUS:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half were placed in Harrison’s fluid for fixation.

SOFT TISSUE EXAMINATION OF THE FETUSES:
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

SKELETAL EXAMINATION OF THE FETUSES:
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were archived individually.

EVALUATION CRITERIA FOR ASSESSING THE FETUSES:
In the present study the glossary of WISE et al. (1997) and its updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (1999) and SOLECKI et al. (2001, 2003):
- Malformation: A permanent structural change that is likely to adversely affect the survival or health.
- Variation: A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development. The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations. All fetal findings were listed in tables according to these classifications.

Statistics:

- DUNNETT's test: Food consumption, body weight, body weight change, corrected body weight gain, carcass weight, weight of the unopened uterus, weight of the placentas and fetuses, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses
- FISHER's exact test: Number of pregnant animals at the end of the study, mortality rate (of the dams) and number of litters with fetal findings
- WILCOXON test: Proportion of fetuses with findings per litter

Indices:

- sex ratio
- The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals / number of fertilized animals) x 100
- The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of corpora lutea – number of implantations) / number of corpora lutea) x 100
- The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of implantations – number of live fetuses) number of implantations) x 100

Historical control data:

yes, period over 5 years

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during the entire study period. Most of the females (18 out of 25) of the high-dose group (1000 mg/kg bw/d) and one female of the mid-dose group (300 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred in the respective animals only within the 2-hour examination interval (i.e. < 2h after treatment). It is considered to be treatment-related, likely as a result of the bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no substance-related or spontaneous mortalities in any females of all test groups (0, 100, 300 or 1000 mg/kg bw/d).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights (BW) and the average body weight gains (BWC) of the low-, mid and high-dose dams (100, 300, 1000 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. In addition, the corrected body weight gain of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) revealed no difference of any biological relevance to the concurrent control group. Moreover, mean carcass weights remained also unaffected by the treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of the dams in test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No necropsy findings which could be attributed to the test substance were seen in any dam. There occurred one spontaneous finding in test groups 0 and 1 (0 and 100 mg/kg bw/d), i.e. dilated renal pelvis. This finding was detected in one control and two low-dose animals and was therefore assessed as incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) in the values calculated for the pre- and the postimplantation losses.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) in conception rate, in the number of resorptions.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) in number of resorptions.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) in conception rate, in the number of viable fetuses.

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:

no effects observed

Description (incidence and severity):

The conception rate reached 96% in the mid-dose group (300 mg/kg bw/d) and 100% in the control, low- and high-dose groups (0, 100 and 1000 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study.

Other effects:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) in the mean number of corpora lutea.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No toxic effects at the highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control group.
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:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control fetuses.

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus with two external malformations was recorded in test group 3 (1000 mg/kg bw/d). The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data. No external variations were recorded. No external unclassified observations were recorded. No unclassified soft tissue observations were recorded.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A number of skeletal malformations were detected in fetuses of test groups 0, 1 and 2 (0, 100 and 300 mg/kg bw/d) affecting the skull and humerus. An association of these malformations to the treatment is not assumed. For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were covered by the historical control data. Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the sternum and ribs and did not show any relation to dosing.

Visceral malformations:

no effects observed

Description (incidence and severity):

No soft tissue malformations were recorded. Three soft tissue variations were detected, i.e. misaligned palatal rugae, dilated renal pelvis and dilated ureter. These variations were neither significantly different from the concurrent control nor dose-dependently altered. Therefore, they were not considered to be biologically relevant

Other effects:

no effects observed

Description (incidence and severity):

The mean placental weights of the low-, mid- and high-dose groups (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control group.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxic effects observed at the highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Total indcidence of external malformations

test group	Dam No, Fetus No, Sex	Findings
0 (0 mg/kg bw)	none
1 (100 mg/kg bw)	none
2 (300 mg/kg bw)	none
3 (1000 mg/kg bw)	94 -10 M	anal atresia, thread-like tail

                                                                             

Total soft tissue variations

		group 0 - 0 mg/kg bw/d	group 1 -100 mg/kg bw/d	group 2 -300 mg/kg bw/d	group 3 -1000 mg/kg bw/d
Litter Fetuses	N N	25 136	24 121	23 108	24 119
fetal incidence	N (%)	2 (1.5)	1 (0.8)	1 (0.9)	2 (1.7)
litter incidence	N (%)	2 (8)	1 (4.2)	1 (4.3)	2 (8.3)
affected fetuses / litter	mean %	1.6	1.0	1.1	1.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Individual fetal skeletal malformations

test group	Dam No, Fetus No, Sex	Findings
0 (0 mg/kg bw)	12-01 F	misshapen tuberositas deltoidea
1 (100 mg/kg bw)	41-07 M	exoccipital fused with 1st cervical arch,misshapen basisphenoid
2 (300 mg/kg bw)	69-13 M71-05 M	severely malformed skull bonesshortened humerus
3 (1000 mg/kg bw)	none

Total fetal skeletal malformations

		group 0 - 0 mg/kg bw/d	group 1 -100 mg/kg bw/d	group 2 -300 mg/kg bw/d	group 3 -1000 mg/kg bw/d
Litter Fetuses	N N	23 119	25 132	25 122	25 130
fetal incidence	N (%)	1 (0.8)	0.0	2 (1.6)	2 (1.5)
litter incidence	N (%)	1 (4.3)	0.0	1 (4.0)	2 (8.0)
affected fetuses / litter	mean %	0.9	0.0	1.3	1.6

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Total fetal skeletal variations

		group 0 - 0 mg/kg bw/d	group 1 -100 mg/kg bw/d	group 2 -300 mg/kg bw/d	group 3 -1000 mg/kg bw/d
Litter Fetuses	N N	25 143	25 132	23 121	24 130
fetal incidence	N (%)	1 (0.7)	1 (0.8)	2 (1.7)	0.0
litter incidence	N (%)	1 (4.0)	1 (4.0)	2 (8.7)	0.0
affected fetuses / litter	mean %	0.6	0.6	1.2	0.0

Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

For a better overview, all skeletal variations with statistically significant differences between the control and the treated groups were compiled in the table below. All incidences were expressed on a fetus per litter basis and any statistically significant differences, which were outside historical control ranges were marked in bold and italics types.

Finding	group 0 - 0 mg/kg bw/d	group 1 -100 mg/kg bw/d	group 2 -300 mg/kg bw/d	group 3 -1000 mg/kg bw/d	HCD Mean %(range)
Incomplete ossification of parietal; unchanged cartilage	2.0	6.8	8.9*	9.0*	10.5(4.5 – 17.1)
Incomplete ossification of thoracic centrum; unchanged cartilage	0.0	2.2	0.7	1.7	1.0(0.0 – 4.8)
Dumbbell ossification of thoracic centrum; unchanged cartilage	0.8	5.5	4.3	5.0	5.2(0.0 – 14.5)
Supernumerarythoracic vertebra	0.8	6.1	5.9*	1.3	4.1(0.8 - 11.0)

HCD = Historical control data; % = per cent * = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

As can be seen from this table the increased incidences of skeletal variations were either not related to the dose or they were inside the historical control range. They are in any case not considered as adverse events.

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 1000 mg/kg bw/d.

Executive summary:

In a prenatal developmental toxicity study, in accordance with GLP and OECD 414, the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Generally, clinical observations including water consumption, food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving 100, 300 or 1000 mg/kg bw/d and controls. Most of the females (18 out of 25) of the high-dose group (1000 mg/kg bw/d) and one female of the mid-dose group (300 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred in the respective animals only within the 2-hour examination interval (i.e. < 2h after treatment). It is considered to be treatment-related, likely as a result of the bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 March 2022 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

Version / remarks:

Version was not specified
The following deviations were noted:
The following animals were administered a partial dose on the listed Day; B0204 and B0216 (Group 3) GD8, B0305 and B0307 (Group 4) GD10, B0217 (Group 3) GD13, B0110 (Group 2) and B0202 (Group 3) GD15, B0203 (Group 3) GD16, B0108 (Group 2), B0204 (Group 3), and B0301 (Group 4) GD17, B0212 (Group 3) on GD18, B0217 (Group 3) on GD20’ B0105 (Group 2) on GD22 and B0013 (Group 1) on GD23. On GD19, animal B0309 (Group 4) was inadvertently not dosed. On GD 28, post-dose observations were performed late for Subgroup 4 animals. On 15 April 2022 (GD 16, 17, 18, or 19, as applicable), post-dose observations were performed late for Group 1 animals. On GD23, Animal B0213 (Group 3) did not have an empty feeder taken. On GD29, Animal B0004 (Group 1) had a macroscopic observation recorded for the uterus. However, this tissue was not collected as required.

Deviations:

yes

Remarks:

see above remarks

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Version was not specified
The following deviations were noted:
The following animals were administered a partial dose on the listed Day; B0204 and B0216 (Group 3) GD8, B0305 and B0307 (Group 4) GD10, B0217 (Group 3) GD13, B0110 (Group 2) and B0202 (Group 3) GD15, B0203 (Group 3) GD16, B0108 (Group 2), B0204 (Group 3), and B0301 (Group 4) GD17, B0212 (Group 3) on GD18, B0217 (Group 3) on GD20’ B0105 (Group 2) on GD22 and B0013 (Group 1) on GD23. On GD19, animal B0309 (Group 4) was inadvertently not dosed. On GD 28, post-dose observations were performed late for Subgroup 4 animals. On 15 April 2022 (GD 16, 17, 18, or 19, as applicable), post-dose observations were performed late for Group 1 animals. On GD23, Animal B0213 (Group 3) did not have an empty feeder taken. On GD29, Animal B0004 (Group 1) had a macroscopic observation recorded for the uterus. However, this tissue was not collected as required.

Deviations:

yes

Remarks:

See above remark

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

The following deviations were noted:
The following animals were administered a partial dose on the listed Day; B0204 and B0216 (Group 3) GD8, B0305 and B0307 (Group 4) GD10, B0217 (Group 3) GD13, B0110 (Group 2) and B0202 (Group 3) GD15, B0203 (Group 3) GD16, B0108 (Group 2), B0204 (Group 3), and B0301 (Group 4) GD17, B0212 (Group 3) on GD18, B0217 (Group 3) on GD20’ B0105 (Group 2) on GD22 and B0013 (Group 1) on GD23. On GD19, animal B0309 (Group 4) was inadvertently not dosed. On GD 28, post-dose observations were performed late for Subgroup 4 animals. On 15 April 2022 (GD 16, 17, 18, or 19, as applicable), post-dose observations were performed late for Group 1 animals. On GD23, Animal B0213 (Group 3) did not have an empty feeder taken. On GD29, Animal B0004 (Group 1) had a macroscopic observation recorded for the uterus. However, this tissue was not collected as required.

Deviations:

yes

Remarks:

See above remark

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Sponsor (batch: WO61290 or OC061290)
- Purity, including information on contaminants, isomers, etc.: 100% (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Not specified
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not specified
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Stable under room temperature and protected from light conditions up to 24 hours and under refrigerated (2 to 8C) and frozen (set to maintain -20  10C) conditions up to 15 days.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Formulated in corn oil, protected from light
- Preliminary purification step (if any): None
- Final concentration of a dissolved solid, stock liquid or gel: 750 mg/mL
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle):


OTHER SPECIFICS: None

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo Global Services Inc
- Age at study initiation: 7 months
- Weight at study initiation: 2818 to 3806 g.
- Fasting period before study: Not specified
- Housing: individually housed in suspended stainless steel cages
- Diet (e.g. ad libitum): Rabbit Diet #2030C
- Water (e.g. ad libitum):ad libitum.
- Acclimation period:3, 4, or 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 to 22
- Humidity (%): 30 to 70
- Air changes (per hr): minimum 10
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: To: 02 April 2022 - 28 April 2022

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item formulations were mixed four times according to the mixing procedure developed by the Laboratory and apportioned for use. Dose concentrations were based on the test item as supplied. Dose formulations were protected from light in a refrigerator, set to maintain 2 to 8C,
until removed for dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on solubility
- Amount of vehicle (if gavage): Dose volume of up to 1 mL/kg
- Lot/batch no. (if required): MKCP9878/MKCQ0649
- Purity: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test item prepared in vehicle control item at concentrations of 10 and 750 mg/mL was evaluated under room temperature and protected from light conditions up to 24 hours and under refrigerated (2 to 8C) and frozen (set to maintain -20 - 10C) conditions up to 15 days.
All homogeneity and concentration verification results met acceptance criteria. The mean percent of target results were within 85 to 115% (90.5 to 93.6%), and the relative standard deviation was not more than 10% and met specifications for concentration verification. No significant absorbance was detected in the control samples.

Details on mating procedure:

Pre-mated females were used

Duration of treatment / exposure:

GD6 - GD28

Frequency of treatment:

Once a day

Duration of test:

GD6 - GD28

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

50 mg/kg bw/day

Remarks:

Low

Dose / conc.:

200 mg/kg bw/day

Remarks:

Mid

Dose / conc.:

750 mg/kg bw/day

Remarks:

High

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for (rat) dam thyroid hormones:
- Time of day for (rat) dam blood sampling:
- Other:

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during the dosing interval at approximately 1 hour post-dose. Detailed observations were made on GD 3, 6, 7, 9, 12, 15, 17, 19, 22, 25, 28, and 29 for each animal. Abnormal findings or an indication of normal were recorded. Unscheduled observations were recorded as they were observed.
- Cage side observations checked in table [No.?] were included. Not stated

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (a.m. and p.m.)

BODY WEIGHT: Yes
- Time schedule for examinations: GD 3, 6, 7, 9, 12, 15, 17, 19, 22, 25, 28, and 29

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # GD29
- Organs examined: Kidney, liver, parathyriod, thyriod, uterus

OTHER: None

Ovaries and uterine content:

The ovaries and uterine content were 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: None

Blood sampling:

- Plasma: No
- Serum: No
- Volume collected: N/A
- Other: None

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter ]
- Skeletal examinations: Yes: [all per litter ]
- Head examinations: Yes: [all per litter ]
- Anogenital distance of all live rodent pups: Not specified

Statistics:

Analysis of variance (ANOVA) and pairwise comparisons were used. Prior to performing the ANOVA, Levene’s test was performed to test for equality of variances between groups.

Only data collected on or after the first day of dosing were analyzed statistically. Data from non-pregnant animals were excluded from statistical analysis.
The pairwise comparisons of interest are:
Group 1 versus Groups 2, 3, and 4
Means and standard deviations were calculated. All statistical tests were evaluated at the
5.0%, 1.0%, and 0.1% probability levels.
Due to system limitations, additional statistical analyses were run but were not reported or used to interpret study data.

Historical control data:

Historical laboratory control data were used.

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related clinical observations were noted. All clinical observations noted in the study appeared infrequently, were noted in controls, or were non-dose responsive; therefore, they were considered not test item related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortalities were noted; all animals survived to their scheduled sacrifice

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related alterations in mean body weight or mean body weight gain were noted for animals administered up to 750 mg/kg/day. Mean body weights and mean body weight gains for test item-treated groups were similar to controls

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related alteration to mean food consumption was noted. Mean food consumption values for test item-treated animals were similar to controls during the entire study (GD 6 to 29).

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related increases in organ weights were noted for the thyroid/parathyroid for animals administered 50, 200, or 750 mg/kg/day compared to controls. However, macroscopic or microscopic correlates to the increased thyroid/parathyroid weights were not noted, thus, they were considered nonadverse.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related macroscopic observations were noted for animals administered up to 750 mg/kg/day. All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for pregnant rabbits; therefore, they were considered not test item related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related microscopic observations were noted in the thyroid/parathyroid for animals administered up to 750 mg/kg/day. All microscopic findings noted in the thyroid/parathyroid were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for pregnant rabbits; therefore, they were considered not test item related.

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: the highest tested dose

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

No test item-related fetal skeletal malformations were noted. All malformations noted were singular incidences, noted with similar incidences with control, and/or noted in a non-dose-responsive manner; thus, they were considered incidental and not test item related.

No test item-related fetal skeletal variations were noted. Sternebra bipartite ossification were noted in animals administered 0, 50, 200, or 750 mg/kg/day. Litter and fetal incidences for animals administered 0, 50, or 200 mg/kg/day were within the Laboratory Historical Control Data ranges (maximum litter incidence of 29% and fetal incidence of 3.89%). Statistically significant increase in litter incidence were noted for animals administered 750 mg/kg/day (32%) compared to concurrent control (5%); however, litter and fetal incidences for animals administered 750 mg/kg/day (32% and 4.36%, respectively) were only slightly outside of the Laboratory Historical Control Data ranges based on percentages. In addition, the number of litters with sternebra bipartite ossification for animals administered 750 mg/kg/day (6 litters) was within the Laboratory Historical Control Data range (max number of litter: 6); therefore, the observed Sternebra bipartite ossification was considered not test item related. All other variations noted were singular incidences, were noted with similar incidences with control, were noted in a non- dose responsive manner, and/or were within the Laboratory Historical Control Data ranges; thus, they were considered incidental and not test item related

Visceral malformations:

no effects observed

Description (incidence and severity):

No test item-related fetal visceral malformations were noted. All malformations noted were singular incidences, were noted in controls, and/or were noted in a non-dose- responsive manner; thus, they were considered incidental and not test item related.

No test item-related fetal visceral variations were noted. Supernumerary adrenal was noted in one fetus from one litter maternally administered 200 mg/kg/day (litter incidence of 5% and fetal incidence of 0.56%) and two fetuses from two litters maternally administered 750 mg/kg/day (litter incidence of 11% and fetal incidence of 1.14%); however, none was noted in controls. The litter and fetal incidences of animals administered 200 mg/kg/day were within the Laboratory Historical Control Data range (maximum litter incidence of 10% and fetal incidence of 1.07%) while the values for animals administered 750 mg/kg/day were only slightly outside of the range based on percentage. However, the number of litters with supernumerary adrenal was within the Laboratory Historical Control Data range (max number of litter: 2); thus, the observations of supernumerary adrenal were considered not test item related. All other variations noted were singular incidences, were noted with similar incidences with control, and/or were noted in a non-dose-responsive manner; thus, they were considered incidental and not test item related.

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: the highest tested dose

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 3: Results of Homogeneity and Concentration Verification Analyses Method Reference: 12795

Interval	Location	Replicate	0 mg/mL		50 mg/mL		200 mg/mL		750 mg/mL
			Actual	% of Target	Actual	% of Target	Actual	% of Target	Actual	% of Target
Day 1	Top	1	-	-	46.8	93.7	-	-	682	90.9
(TA 1)		2	-	-	44.4	88.9	-	-	692	92.3
	Middle	1	DNS	-	47.9	95.8	182	90.8	691	92.1
		2	-	-	47.6	95.2	182	91.0	701	93.5
		3	-	-	-	-	184	92.2	-	-
	Bottom	1	-	-	45.5	91.1	-	-	702	93.6
		2	-	-	45.0	90.0	-	-	701	93.5
		Mean	-	-	-	92.4	-	91.3	-	92.6
		RSD (%)	-	-	-	3.1	-	0.9	-	1.2
Last Day	Middle	1	DNS	-	45.5	91.1	178	88.9	695	92.7
(TA 4)		2	-	-	46.3	92.5	181	90.6	700	93.3
		3	-	-	46.7	93.4	184	91.9	711	94.9
		Mean	-	-	-	92.3	-	90.5	-	93.6
		RSD (%)	-	-	-	1.3	-	1.7	-	1.2

- = Not applicable; DNS = Detection not significant; RSD = Relative standard deviation

 

Table 4. Summary of Clinical Observations

Category Observation		0 mg/kg	50 mg/kg	100 mg/kg	750 mg/kg
No remarkable observations		20	20	20	20
Behavior - other	aggression to handle	0	1	1	0
	sensitivity to touch, increased	0	0	1	0
	struggled during dosing	0	1	0	0
Discharge	source known, mouth, red	1	0	0	0
	source known, vulva, red	1	0	0	0
Pelage	abnormal colour, abdomen, right, red	0	0	1	0
	hair loss, abdomen, right	0	0	1	0
	hair loss, foot, hind right	0	0	1	0
Skin	laceration, mouth	0	1	0	0
	palpable mass, vulva, medium	0	0	0	1
	scab, ear, left	0	0	0	1
	scab, foot, hind right	1	0	0	0
	scab, mouth	0	1	1	0
	sore, abdomen, right, dry	0	0	1	0
Teeth	broken, lower left	0	0	0	1
	broken, upper left	1	0	0	0

 

Table 5. Summary of Body Weight

Group/ mg/kg/day	Phase	GE
	Day	3	6	7	9	12	15	17	19	22	25	28
0	Mean	3304	3335	3336	3348	3386	3474	3480	3505	3526	3585	3614
	SD	221.6	218.4	220.2	224.8	220.1	243.4	253.6	262.4	267.7	253.3	251.5
	N	20	20	20	19	20	20	20	20	20	20	20
50	Mean	3324	3351	3337	3368	3387	3466	3493	3496	3533	3591	3631
	SD	220.2	207.6	203.1	208.1	224.3	242.8	247.8	253.4	245.8	239.6	258.0
	N	18	18	17	18	18	18	18	18	18	18	18
200	Mean	3315	3352	3359	3368	3383	3437	3454	3473	3510	3574	3597
	SD	186.3	170.7	177.3	185.6	190.0	225.2	232.7	227.0	231.2	239.4	243.1
	N	20	20	20	20	20	20	20	20	20	20	20
750	Mean	3325	3365	3380	3384	3407	3493	3500	3519	3545	3608	3636
	SD	215.1	208.1	212.0	221.8	234.2	256.5	259.1	263.3	224.7	215.7	190.2
	N	19	19	19	19	19	19	19	19	19	19	19
Statistics		X1	A	A	A	A	A	A	A	A	A	A

GE = Gestation; X1 = No analysis required; A = ANOVA and Dunnett's

 

Table 6. Summary of Body Weight Change

Group/ mg/kg/day	Phase	GE
	Day	3-6	6-7	7 - 9	9 - 12	12 - 15	15 -17	17 -19	19 - 22	22 - 25	25 - 28	28 - 29	6 - 29
0	Mean	31	1	23	27	88	6	26	20	59	29	16	294
	SD	30.7	26.0	33.1	36.4	45.9	45.3	56.3	49.2	50.5	50.0	53.4	126.1
	N	20	20	19	19	20	20	20	20	20	20	20	20
50	Mean	27	1	17	19	78	27	3	36	58	40	22	302
	SD	38.7	37.8	28.5	38.4	65.5	41.9	24.0	37.5	59.9	69.9	21.1	153.6
	N	18	17	17	18	18	18	18	18	18	18	18	18
200	Mean	37	7	9	15	54	17	19	36	64	23	17	262
	SD	43.7	29.2	28.9	48.0	73.1	33.1	27.1	51.2	40.2	31.4	28.4	147.9
	N	20	20	20	20	20	20	20	20	20	20	20	20
750	Mean	40	15	3	23	86	7	20	26	63	27	9	279
	SD	45.5	35.8	22.8	34.8	52.7	40.0	43.9	59.5	61.6	48.4	29.4	126.8
	N	19	19	19	19	19	19	19	19	19	19	19	19
Statistics		X1	A	A	A	A	A	A	A	A	A	AT	A

GE = Gestation, X1 = No analysis required;  A = ANOVA and Dunnett's; T = Rank-transformed data

Table 6. Summary of Food Consumption

Group/ mg/kg/day	Phase	GE
	Day	3-4	4-5	5-6	6-7	7-8	8-9	9-10	10-11	11-12	12-13	13-14	14-15	15-16	16-17	17-18	18-19		19-20	20-21	21-22	22-23	23-24	24-25	25-26	26-27	27-28	28-29	6-29
0	Mean	117	138	137	129	129	131	126	123	115	98	108	117	113	105	103	108		108	115	112	106	85	81	68	76	77	81	2424
	SD	37.4	15.2	18.8	23.5	24.1	22.3	26.7	26.5	26.8	38.2	32.0	33.0	37.5	44.0	48.2	41.8		40.6	33.5	35.7	33.4	41.4	31.1	36.0	32.8	33.7	38.1	453.3
	N	20	20	20	20	20	20	20	20	20	20	20	20	20	20	20	20		20	20	20	20	20	20	20	20	19	20	19
50	Mean	118	141	144	135	137	135	135	118	119	126	114	113	111	109	99	102		107	117	113	108	101	94	81	84	87	95	2539
	SD	41.5	17.0	12.6	27.3	16.6	19.7	17.0	36.6	32.8	27.0	32.2	42.6	38.9	43.4	50.3	45.8		41.1	35.8	33.4	36.7	34.3	40.0	36.1	37.5	36.6	30.	478.1
	N	18	18	18	18	18	18	18	18	18	18	18	18	18	18	18	18		18	18	18	18	18	18	18	18	18	18	18
200	Mean	122	143	144	138	141	131	128	120	122	111	107	103	110	101	100	110		109	111	115	104	106	100	90	88	87	84	2607
	SD	40.2	15.5	14.9	21.0	14.5	23.0	20.9	21.2	28.3	36.9	36.5	53.8	42.5	50.0	45.5	36.7		29.1	31.2	31.2	41.4	30.9	27.7	34.9	29.1	31.4	33.3	438.6
	N	20	20	20	20	20	20	20	20	20	20	20	20	19	20	20	20		20	20	20	19	20	20	20	20	20	20	18
750	Mean	120	143	141	133	127	124	117	110	114	109	111	110	107	122	112	117		120	117	116	105	96	96	85	92	95	97	2567
	SD	50.5	18.1	24.7	29.3	24.3	26.3	31.9	26.9	33.6	33.0	40.2	31.3	35.3	21.1	35.6	35.3		37.5	39.9	35.4	40.9	34.7	31.4	32.8	28.5	34.3	29.1	412.7
	N	19	19	19	19	19	19	19	19	19	19	19	19	19	18	19	19		19	19	19	19	19	19	19	19	19	19	18
Statistics		X1	X1	X1	A	A	A	AT	A	A	A	AT	AT	A	AT	A	A	A		A	A	A	A	A	A	A	A	A	A

GE = Gestation, X1 = No analysis required;  A = ANOVA and Dunnett's; T = Rank-transformed data

Table 7. Summary of Reproductive Performance

Treatment	0 mg/kg	50 mg/kg	200 mg/kg	750 mg/kg
Total Females	20	20	20	20
Pregnant Females	20	18	20	19
Non-pregnant Females	0	2	0	1
Pregnant with Total Litter Loss	0	0	0	0
Females with Live Fetuses	20	18	20	19

 

Table 8. Summary of Cesarean Section Data

Treatment		0 mg/kg	50 mg/kg	200 mg/kg	750 mg/kg
Number of females pregnant at cesarean section (n)		20	18	20	19
Corpora Lutea	(n)	20	18	20	19
	Mean	10.2	10.6	9.9	9.5
	SD	2.08	2.30	2.40	1.71
Implantation Sites	(n)	20	18	20	19
	Mean	9.2	9.1	8.6	8.9
	SD	1.85	2.08	2.46	1.94
Preimplantation Loss	(n)	20	18	20	19
	Mean	1.0	1.5	1.4	0.6
	SD	1.15	1.42	1.27	0.90
Preimplantation Loss (%)	(n)	20	18	20	19
	Mean	8.76	13.65	13.37	6.75
	SD	10.146	12.203	13.101	10.301
Early Resorptions	(n)	20	18	20	19
	Mean	0.3	0.2	0.3	0.1
	SD	0.44	0.43	0.64	0.46
Late  Resorptions	(n)	20	18	20	19
	Mean	0.1	0.1	0.2	0.1
	SD	0.31	0.47	0.37	0.32
Total Resorptions	(n)	20	18	20	19
	Mean	0.4	0.3	0.4	0.2
	SD	0.49	0.59	0.75	0.71
Dead Fetuses	(n)	20	18	20	19
	Mean	0.0	0.0	0.0	0.0
	SD	0.00	0.00	0.00	0.00
Postimplantation Loss	(n)	20	18	20	19
	Mean	0.4	0.3	0.4	0.2
	SD	0.49	0.59	0.75	0.71
Postimplantation Loss (%)	(n)	20	18	20	19
	Mean	4.00	3.40	6.40	2.50
	SD	5.750	6.288	13.652	8.780
Live Fetuses	(n)	20	18	20	19
	Mean	8.9	8.8	8.2	8.7
	SD	1.95	2.02	2.81	2.11
Gravid Uterine Weight	(n)	20	18	20	19
	Mean	502.37	511.12	462.87	504.67
	SD	88.749	107.956	113.869	85.919
Corrected Body Weight (Carcass Weight)	(n)	20	18	20	19
	Mean	3106.03	3131.22	3138.68	3123.75
	SD	238.141	249.427	241.924	184.301
Corrected Weight Change	(n)	20	18	20	19
	Mean	-197.72	-192.56	-176.42	-201.51
	SD	121.194	158.090	156.240	122.499
Total Weight Change (Weight Change)	(n)	20	18	20	19
	Mean	304.65	318.56	286.45	303.16
	SD	130.035	156.401	169.616	131.514

 

Table 9. Summary of Mean Fetal Data

Treatment		0 mg/kg	50 mg/kg	200 mg/kg	750 mg/kg
Number of Females with Live Fetuses (n)		20	18	20	19
Mean Number of Male Fetuses per Litter	(n)	20	18	20	19
	Mean	4.7	4.3	4.0	4.4
	SD	2.23	1.64	2.01	1.84
Mean Number of Female Fetuses per Litter	(n)	20	18	20	19
	Mean	4.2	4.5	4.2	4.3
	SD	1.46	1.62	2.02	1.73
% Male Fetuses	(n)	20	18	20	19
	Mean	51.8	48.4	48.1	51.1
	SD	18.86	16.94	16.70	16.93
Mean Fetal Weight	(n)	20	18	20	19
	Mean	40.23	40.25	40.91	41.01
	Adj Mean	40.54	40.47	40.31	41.10
	SD	3.257	3.878	5.546	4.890
Mean Weight - Male Fetuses (n)	(n)	19	18	20	19
	Mean	40.97	40.79	41.78	41.33
	Adj Mean	41.45	40.96	41.12	41.38
	SD	2.990	4.168	5.962	4.863
Mean Weight - Female Fetuses (n)	(n)	20	18	20	19
	Mean	39.46	40.11	40.30	40.36
	Adj Mean	39.77	40.33	39.70	40.46
	SD	3.913	4.140	5.769	5.393
	SD	5.750	6.288	13.652	8.780
Live Fetuses	(n)	20	18	20	19
	Mean	8.9	8.8	8.2	8.7
	SD	1.95	2.02	2.81	2.11
Gravid Uterine Weight	(n)	20	18	20	19
	Mean	502.37	511.12	462.87	504.67
	SD	88.749	107.956	113.869	85.919
Corrected Body Weight (Carcass Weight)	(n)	20	18	20	19
	Mean	3106.03	3131.22	3138.68	3123.75
	SD	238.141	249.427	241.924	184.301
Corrected Weight Change	(n)	20	18	20	19
	Mean	-197.72	-192.56	-176.42	-201.51
	SD	121.194	158.090	156.240	122.499
Total Weight Change (Weight Change)	(n)	20	18	20	19
	Mean	304.65	318.56	286.45	303.16
	SD	130.035	156.401	169.616	131.514

 

Table 10. Summary of Fetal Observations - Malformations

Malformation	Tissue observed	Numbers observed	0 mg/kg	50 mg/kg	200 mg/kg	750 mg/kg	Stats
External Malformations	Eye	Number of maternal female / fetuses	20/179	20/159	20/163	20/167
		Number  examined litter/fetus	20/177	18/158	20/163	19/165
	M-eye open	Examined litter/fetus	20/177	18/158	20/163	19/165
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
	General	Examined Litter/Fetus	20/177	18/158	20/163	19/165
	M-gastroschisis		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
	Head	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-domed		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.56	0.00	X2
	M-exencephaly		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
	M-micrognathia		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
	Limb	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-hyperflexion		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
	Paw	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-brachydactyly		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
	Tail	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-tail - bent		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
Fresh Visceral Malformations and Variations	Adrenal	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-adrenal - malpositioned		1/2	1/1	0/0	0/0
		% Litter	5	6	0	0	F+
		%fetal	1.25	0.51	0.00	0.00	X2
	Blood Vessel	Examined litter/fetus	20/176	18/158	20/163	19/165
	M-aortic arch - dilated		0/0	0/0	0/0	1/1
		% Litter	0	0	0	5	F+
		%fetal	0.00	0.00	0.00	0.58	X2
	M-ductus arteriosis - malpositioned		0/0	0/0	0/0	1/1
		% Litter	0	0	0	5	F+
		%fetal	0.00	0.00	0.00	0.58	X2
	M-pulmonary artery - malpositioned		0/0	0/0	0/0	1/1
		% Litter	0	0	0	5	F+
		%fetal	0.00	0.00	0.00	0.58	X2
	M-pulmonary trunk - atretic		0/0	0/0	0/0	1/1
		% Litter	0	0	0	5	F+
		%fetal	0.00	0.00	0.00	0.58	X2
	Brain	Examined litter/fetus	20/89	18/77	20/80	19/83
	M-internal hydrocephaly		0/0	0/0	2/2	0/0
		% Litter	0	0	10	0	F+
		%fetal	0.00	0.00	2.25	0.00	X2
	Gall Bladder	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-gall Bladder absent		0/0	1/1	0/0	0/0
		% Litter	0	6	0	0	F+
		%fetal	0.00	0.79	0.00	0.00	X2
	Heart	Examined litter/fetus	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-ventricular chamber -absent			0/0	0/0	0/0	1/1
		% Litter	% Litter	0	0	0	5
		%fetal	%fetal	0.00	0.00	0.00	0.58
	Spleen	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-microsplenia		1/1	1/1	0/0	0/0
		% Litter	5	6	0	0	F+
		%fetal	0.63	0.51	0.00	0.00	X2
	Adrenal	Examined litter/fetus	20/177	18/158	20/163	19/165
	V-adrenal - cyst.		5/5	1/1	2/2	2/3
		% Litter	25	6	10	11	F+
		%fetal	2.55	0.93	1.34	1.79	X2
	V-adrenal - discolored.		1/1	0/0	0/0	0/0
		% Litter	5	0	0	0	F+
		%fetal	0.38	0.00	0.00	0.00	X2
	V-adrenal - supernumerary.		0/0	0/0	1/1	2/2
		% Litter	0	0	5	11	F+
		%fetal	0.00	0.00	0.56	1.14	X2
	Blood Vessel	Examined litter/fetus	20/176	18/158	20/163	19/165
	V-carotid artery - Supernumerary branch.		0/0	0/0	0/0	1/1
		% Litter	0	0	0	5	F+
		%fetal	0.00	0.00	0.00	0.75	X2
	V-renal vein -supernumerary branch.		0/0	1/1	0/0	1/1
		% Litter	0	6	0	5	F+
		%fetal	0.00	0.93	0.00	0.53	X2
	V-vena cava - branching variation.		3/4	1/1	1/1	1/1
		% Litter	15	6	5	5	F+
		%fetal	2.01	0.69	0.50	0.53	X2
	Gall Bladder	Examined litter/fetus	20/177	18/158	20/163	19/165
	V-gall bladder – cyst.		0/0	1/1	0/0	0/0
		% Litter	0	6	0	0	F+
		%fetal	0.00	0.51	0.00	0.00	X2
	V-gall bladder- small.		1/1	0/0	1/1	0/0
		% Litter	5	0	5	0	F+
		%fetal	0.51	0.00	0.38	0.00	X2
	Liver	Examined litter/fetus	20/177	18/158	20/163	19/165
	V-liver lobe discolored		1/1	0/0	0/0	0/0
		% Litter	5	0	0	0	F+
		%fetal	0.50	0.00	0.00	0.00	X2
V-liver lobe supernumerary		1/1	3/4	2/2	2/3
	% Litter	5	17	10	11	F+
	%fetal	0.63	2.89	1.10	1.83	X2
Lung	Examined litter/fetus	20/177	18/158	20/163	19/165
V-intermediate lobe -          absent		4/9	0/0	0/0	0/0
	% Litter	20	17	0	26	F+
	%fetal	5.26	4.17	0.00	4.09	X2
Ovary	Examined litter/fetus	20/83	18/81	20/84	19/81
V-ovary - cyst.		0/0	1/1	1/2	0/0
	% Litter	0	6	5	0	F+
	%fetal	0.00	1.11	2.50	0.00	X2
V-ovary- discolored		0/0	1/1	1/2	0/0
	% Litter	5	0	0	0	F+
	%fetal	1.00	0.00	0.00	0.00	X2
Spleen	Examined litter/fetus	20/177	18/158	20/163	19/165
V-spleen - misshapen		1/2	3/3	0/0	0/0
	% Litter	5	17	0	0	F+
	%fetal	1.00	2.36	0.00	0.00	X2
V-spleen - supernumerary		14/27	12/30	10/20	12/24
		70	67	50	63	F+
		15.15	18.90	14.19	15.31	X2
Stomach	Examined litter/fetus	20/177	18/158	20/163	19/165
V-stomach - distended		0/0	1/1	0/0	0/0
	% Litter	0	6	0	0	F+
	%fetal	0.00	0.69	0.00	0.00	X2
Thymus	Examined litter/fetus	20/177	18/158	20/163	19/165
V-thymus - discolored		0/0	1/1	2/3	0/0
	% Litter	0	6	10	0	F+
	%fetal	0.00	0.69	1.81	0.00	X2
Ureter	Examined litter/fetus	20/177	18/158	20/163	19/165
V-ureter - retrocaval		0/0	1/1	0/0	4/5
	% Litter	0	6	0	21	F+
	%fetal	0.00	0.69	0.00	2.82	K
Malformation in Fetal Skeletal Observations	Forelimb	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-metacarpal absent		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
	Rib	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-rib - interrupted		1/1	0/0	1/1	1/1
		% Litter	5	0	5	5	F+
		%fetal	0.56	0.00	0.56	0.48	X2
	M-ribs - fused		1/1	0/0	1/1	0/0
		% Litter	5	0	5	0	F+
		%fetal	0.63	0.00	0.56	0.00	X2
	Skull	Examined litter/fetus	20/89	18/77	20/80	19/83
	M-all skull structures - misshapen		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	1.00	0.00	X2
	M-interparietal - absent		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	1.00	0.00	X2
	M-presphenoid - absent		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	1.00	0.00	X2
	Sternebra	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-sternebra - fused		5/6	1/1	1/1	0/0
		% Litter	25	6	5	0	F+
		%fetal	3.97	0.62	1.43	0.00	K
	M-sternebra - supernumerary		1/1	1/1	0/0	0/0
		% Litter	5	6	0	0	F+
		%fetal	1.00	0.51	0.00	0.00	X2
	M-sternebra - wide		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
	Vertebra - lumbar	Examined litter/fetus	20/177	18/158	20/163	19/165
	M- lumbar vertebra absent		1/1	1/3	0/0	0/0
		% Litter	5	6	0	0	F+
		%fetal	0.56	2.38	0.00	0.00	X2
	Vertebra – lumbar centrum	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-lumbar centrum - hemicentric		0/0	1/1	1/1	1/1
		% Litter	0	6	5	5	F+
		%fetal	0.00	0.51	0.56	0.75	X2
	M-lumbar centrum - misaligned		0/0	0/0	0/0	1/1
		% Litter	0	0	0	5	F+
		%fetal	0.00	0.00	0.00	0.75	X2
	Vertebra - thoracic	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-thoracic vertebra - misaligned		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.56	0.00	X2
	Vertebra – thoracic centrum	Examined litter/fetus	20/177	18/158	20/163	19/165
	M-thoracic centrum - fused		1/1	0/0	0/0	0/0
		% Litter	5	0	0	0	F+
		%fetal	0.38	0.00	0.00	0.00	X2
	M-thoracic centrum - hemicentric		1/1	0/0	0/0	0/0
		% Litter	5	0	0	0	F+
		%fetal	0.38	0.00	0.00	0.00	X2
	M-thoracic centrum - misaligned		0/0	1/1	0/0	0/0
		% Litter	0	5	0	0	F+
		%fetal	0.00	0.51	0.00	0.00	X2
Variation in Fetal Skeletal Observations	Forelimb	Examined litter/fetus	20/177	18/158	20/163	19/165
	V-phalanx -                  unossified.		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
	Hindlimb	Examined litter/fetus	20/177	18/158	20/163	19/165
	V-metatarsal - incomplete ossification.		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
	V-phalanx -                  unossified.		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	0.50	0.00	X2
	V-talus - unossified		0/0	0/0	2/2	0/0
		% Litter	0	0	10	0	F+
		%fetal	0.00	0.00	0.86	0.00	X2
	Rib	Examined litter/fetus	20/177	18/158	20/163	19/165
	V-rib - detached		7/10	6/6	2/2	2/5
		% Litter	35	33	15	11	F+
		%fetal	5.24	3.43	1.55	3.31	K
	V-rib – incomplete ossification		0/0	1/1	0/0	0/0
		% Litter	0	5	0	0	F+
		%fetal	0.00	0.79	0.00	0.00	X2
	Skull	Examined litter/fetus	20/177	18/158	20/163	19/165
	V-frontal - incomplete ossification		1/1	0/0	0/0	0/0
		% Litter	5	0	0	0	F+
		%fetal	0.71	0.00	0.00	0.00	X2
	V-hyoid - bent		1/1	1/1	0/0	4/5
		% Litter	5	6	0	24	F+
		%fetal	0.71	1.11	0.00	6.89	X2
	V-hyoid - unossified		0/0	0/0	2/3	0/0
		% Litter	0	0	10	0	F+
		%fetal	0.00	0.00	3.93	0.00	X2
	V-interparietal - incomplete ossification.		1/3	0/0	0/0	0/0
		% Litter	5	0	0	0	F+
		%fetal	2.14	0.00	0.00	0.00	X2
	V-nasal/frontal - isolated ossification site		0/0	0/0	1/1	0/0
		% Litter	0	0	5	0	F+
		%fetal	0.00	0.00	1.25	0.00	X2
	V-parietal - incomplete ossification.		1/3	0/0	0/0	0/0
		% Litter	5	0	0	0	F+
		%fetal	2.14	0.00	0.00	0.00	X2
	V-parietal/interparietal - Isolated ossification site		0/0	1/1	0/0	2/2
		% Litter	0	6	0	11	F+
		%fetal	0.00	1.85	0.00	2.19	X2
	V-zygomatic arch - Incomplete ossification.		1/1	0/0	0/0	0/0
		% Litter	5	0	0	0	F+
		%fetal	0.71	0.00	0.00	0.00	X2
	Sternebrae	Examined litter/fetus	20/177	18/158	20/163	19/165
	V-sternebra – asymmetric ossification		0/0	1/1	1/1	0/0
		% Litter	0	6	5	0	F+
		%fetal	0.00	0.51	0.50	0.00	X2
	V-sternebra – bipartite ossification.		1/1	4/5	5/6	6/7
		% Litter	5	22	25	32	F+
		%fetal	0.42	2.95	3.52	4.36	K
	V-sternebra – incomplete ossification.		6/12	7/47	6/9	7/12
		% Litter	20	39	30	37	F+
		%fetal	6.94	9.13	5.04	7.17	K
	V-sternebra supernumerary site(s)		4/5	2/2	1/4	5/8
		% Litter	20	11	5	26	F+
		%fetal	2.99	1.41	2.50	4.68	K
	V-sternebra – unossification.		12/28	8/22	10/30	13/34
		% Litter	60	44	50	68	F+
		%fetal	17.16	12.44	17.07	21.06	K
	Supernumerary Rib	Examined litter/fetus	20/177	18/158	20/163	19/165
	V-Cervical rib - unossification.		0/0	0/0	1/1	0/0
% Litter		0	0	5	0	F+
%fetal		0.00	0.00	0.63	0.00	X2
V-supernumerary rib present		18/89	17/68	19/85	18/101
	% Litter	90	94	95	95	F+
	%fetal	47.88	46.90	52.29	61.93	K
Vertebra - caudal	Examined litter/fetus	20/177	18/158	20/163	19/165
V-caudal vertebra - misaligned.		0/0	1/1	0/0	0/0
	% Litter	0	6	0	0	F+
	%fetal	0.00	0.51	0.00	0.00	X2
V-caudal vertebra - unossification.		2/2	3/4	0/0	0/0
	% Litter	10	17	0	0	F+
	%fetal	1.13	2.02	0.00	0.00	X2
Vertebra - lumbar	Examined litter/fetus	20/177	18/158	20/163	19/165
V-lumbar vertebra - supernumerary		2/2	0/0	0/0	4/4
	% Litter	10	0	0	21	F+
	%fetal	0.94	0.00	0.00	2.47	K

F+ = Fisher's exact (upper tail); X2 = Not analyzed (too few distinct values) Class; V = Variation; K = Kruskal-Wallis and Wilcoxon

Conclusions:

Pregnant rabbits were administered vehicle control item or 50, 200, or 750 mg/kg/day Lankroflex ED6 by oral gavage during the period of gestation (GD 6 through 28). Based on the study results the no observed adverse effect level (NOAEL) was determined to be 750 mg/kg/day.

Executive summary:

Lankroflex ED6 (Fatty acids, C14-22, 2-ethylhexyl esters, epoxidized) was administered by oral gavage to 20 groups of pregnant rabbits during the period of gestation (Gestation Days [GD] 6 through 28) at doses of 0, 50, 200, 750 mg/kg bw/day. Assessment of toxicity was based on mortality, clinical observations, body weights, food consumption, necropsy and cesarean section findings, and organ weights.
No mortalities were noted; all animals survived to their scheduled sacrifice. No test item-related clinical observations were noted.
No test item-related alterations in mean body weight or mean food consumption were noted for animals administered up to 750 mg/kg/day. Mean body weights and mean food consumption for test item administered groups were similar to controls.
No test item-related effects on reproductive performance were noted. All pregnant animals had live fetuses. No test item-related effects on cesarean section parameters were noted. No test item-related macroscopic observations were noted.
Test item-related, nonadverse increases in organ weights were noted for the thyroid/parathyroid for animals administered 50, 200, or 750 mg/kg/day compared to controls. No test item-related microscopic observations were noted in the thyroid/parathyroid for animals administered up to 750 mg/kg/day.
No fetal external variations were noted. No test item-related fetal external malformations, fetal visceral malformations or variations, or fetal skeletal malformations or variations were noted.
In conclusion, pregnant rabbits were administered vehicle control item or 50, 200, or 750 mg/kg/day Lankroflex ED6 by oral gavage during the period of gestation (GD 6 through 28). No mortality was noted. No test item-related clinical observations, effects on mean maternal body weight or food consumption, effects on reproductive performances, effects on cesarean section parameters, macroscopic observations, or fetal evaluation findings were observed at any dose. Test item-related, nonadverse increases in organ weights were noted for the thyroid/parathyroid for animals administered 50, 200, or 750 mg/kg/day compared to controls. Based on these results, maternal and embryo-fetal developmental no observed adverse effect level (NOAEL) was determined to be 750 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1 (reliable without restriction)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In an OECD 414 using the 'target substance' in rabbits and an OECD 414 using the read-across 'source substance'  in rats (second species), in both studies the test item was well tolerated by the dams at all the dose levels and was neither embryonic or teratogenic. The No Observable Adverse Effect Level is defined as 1000 mg/kg bw/day in terms of maternotoxic effects and embryofetal development. 

Justification for classification or non-classification

Based on the data available, no classification for reproductive toxicicty is required in the absence of any indication of reproductive toxicity under the CLP Regulation (EC) No. 1272/2008.