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

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

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.

Test guideline 2

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.

Test guideline 3

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

Test material

Test material information

1

Reference substance name:

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

EC Number:

305-962-8

EC Name:

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

Cas Number:

95370-96-0

Molecular formula:

Not necessary, substance is a UVCB

IUPAC Name:

Fatty acids, C14-22, 2ethylhexylesters, epoxidized

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

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control (Corn oil)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

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.

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Any other information on results incl. tables

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

 

 

 

 

 

 

Applicant's summary and conclusion

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.