Ethyl 3,5-dichloro-4-hexadecyloxycarbonyloxybenzoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Oct-27 Nov 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study. No gross examination of the pups was performed after sacrifice.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

no gross examination of pups was performed after sacrifice

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovak National Accreditation Service, Bratislava, Slovak Republic

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

404-740-9

EC Name:

-

Cas Number:

115895-09-5

Molecular formula:

C26H40Cl2O5

IUPAC Name:

ethyl 3,5-dichloro-4-{[(hexadecyloxy)carbonyl]oxy}benzoate

Details on test material:

- Name of test material (as cited in study report): AF-366LM
- Purity test date: 30 Sep 2009
- Lot/batch No.: 4030/PF
- Storage condition of test material: dry area, well ventilated, protect against electrostatic discharge, keep packaging tightly closed

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Velaz Praha, Czech Republic
- Age at study initiation: adult
- Weight at study initiation: (P) Males: 266.1 g (mean); Females: 172.71 g (mean)
- Housing: the animals were kept in cages with bedding (wood shavings). During the pre-mating period 4 rats/sex/cage; during the mating period 1 male and 1 female per cage; during the gestation period pregnant females were caged individually; during the lactation period females were caged individually together with the offspring; after the mating procedure males were returned to the original cages, 4 males per cage.
- Diet: conventional laboratory diet (MP-OŠ-06, BIOFER, 06901 Snina, Slovak Republic), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 Oct 2010 To: 01 Nov 2010 (males), 12-27 Nov 2010 (females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The solution with AF-366 was prepared daily before application and administered within 2 hours. Concentrations of 6 mg/mL, 30 mg/mL and 150 mg/mL in olive oil were prepared by mixing an accurately weighed amount of AF-366 with olive oil. The solution is stable up to 24 hours at laboratory temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance is not soluble in water, therefore olive oil was selected
- Concentration in vehicle: 6, 30 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 100

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 14 days, until proof of pregnancy
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility was considered
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Twice during the study period, approximately 10 mL of the prepared stock solutions (6, 30 and 150 mg/mL) was sampled for analysis. The precision, homogeneity and stability of the formulation was analysed against standard samples by HPLC before use. The precision and homogeneity met the requirements of 90-110% of the nominal concentration (relative SD ≤ 3%). The stability after 24 hours at room temperature was acceptable; as the mean values measured at 24 hours were shown to be within ±5% of the values measured at 0 hours.

Duration of treatment / exposure:

Males: 28 days
Females: up to 56 days (14 days before the mating period, up to 14 days during the mating period, 22–24 days during the gestation period, and 4 days during the lactation period)

Frequency of treatment:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study: adult

Doses / concentrations

Remarks:

Doses / Concentrations:
30, 150 and 750 mg/kg bw/day
Basis:
nominal conc.
analysis of the stock solutions show the actual concentration to be 90-110% of the nominal concentration

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: daily, 1 hour after administration
- Cage side observations included: mortality, clinical signs (piloerection, alopecia, dyspnoea, salivation, smooth stool, diarrhoea, tremors, spasms, hyperactivity, lethargy, cannibalism, aborts, moribund state)

DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: yes
- Time schedule for examinations: the animals were weighed on the first day of dosing, then weekly thereafter, and at study termination. During the gestation period, females were weighed on Day 0, 7, 14, 20; within 24 hours of parturition (Day 1 post-partum); and on Day 4 post-partum

FOOD CONSUMPTION: the food consumption was measured weekly

WATER CONSUMPTION: no

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight. The litters were weighed 24 hours after the birth and on day 4 post-partum.

GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals were sacrificed after 28 days of treatment
- Maternal animals: all surviving females with litters were sacrificed on Day 5 post-partum; all surviving non-pregnant females were sacrificed 24 days after the mating period

GROSS NECROPSY
- Gross necropsy consisted of: external and internal macroscopic examination of gross pathological changes, including the reproductive organs and target organs (kidneys). All the animals that died during the study period and the animals sacrificed at the end of the study were examined. The liver, kidneys (target organ), testes, epididymes, uterus and ovaries were weighed and the corpora lutea and implantation sites were counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
A histopathologic examination was performed on the animals in the control and highest dose group. Samples of the liver, kidneys (target organ), testes, epididymes, uterus, ovaries, prostate (including coagulating glands) and mammary gland were fixed in formaldehyde and examined microscopically.

Postmortem examinations (offspring):

SACRIFICE
- The surviving offspring were sacrificed on day 5 post-partum

Statistics:

The results (body weight, litter weight, absolute organ weight, food consumption, number of corpora lutea and implantation sites, length of pregnancy, number and sex of pups, relative organ weight) were statistically evaluated by the statistical programme Statgraphics. To identify the homogeneity of the groups Bartlett’s test was used. In the case of homogeneity a One-Way Analysis of variance with consecutive Multiple-Ranges tests was used. In the case of non-homogeneity and for the length of pregnancy Kruskal-Wallis One Way Analysis was applied.

Reproductive indices:

The following reproductive indices were calculated: fertility index and gestation (pregnancy) index.

Fertility index = (number of females with implants / number of females mated) x 100

Gestation index = (number of females delivering live pups / number of females with evidence of pregnancy) x 100

Offspring viability indices:

The following indices were calculated: live birth index and viability (survival) index.

Live birth index = (number of live offspring / number of offspring delivered) x 100
Viability (survival) index = (number of live offspring on lactation day 4/number of live offspring delivered) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

alopecia (non-adverse)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

150 and 750 mg/kg bw/day, females: reduced food consumption week 2 of gestation period (non-adverse)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

150 and 750 mg/kg bw/day, females: reduced food consumption week 2 of gestation period (non-adverse)

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
0/12, 1/12, 2/12 and 2/12 females in the control, low-, mid- and high-dose groups died during the study period (Day 43, 41, 44, 40 and 42, respectively). The deaths were not substance-related.
One male had alopecia on study Day 7-27, while in total 5 females had alopecia for 7-15 days in the period from Day 29-54. This is a normal observation in rats during a repeated dose study and is not substance-related.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The food consumption in the treatment groups were comparable to the control group for males. In the female mid- and high-dose groups, food consumption during the gestation period was statistically significantly reduced in week 2 (see Table 1 and 2). This effect is considered to be incidental as the intake was comparable to the control group the rest of the study period. The body weight of all male groups were comparable. In high-dose females the body weight was slightly, but not statistically, reduced during the whole period from study day 0 until the end of the gestation period. This is not treatment-related, but rather due to the random selection of rats for the groups prior to study start.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test substance was applied by gavage, ensuring an accurate dosing. The concentration of the stock solution was verified analytically prior to dosing.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no statistically significant difference in reproductive performance, measured as pregnancy rate (fertility index) and gestation index, between the control group and treatment groups (see Table 3).

On Day 8 of the mating period, 2 males in the control group and 2 males in the 150 mg/kg bw/day group were replaced by other males, as no evidence of a successful mating had been observed yet. For one of the apparently non-pregnant female in each of the control and 150 mg/kg bw/day groups, it became evident later that mating with the original male was successful.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no treatment-related effects on the organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
In the low-dose female that died (after delivery), autolysis was evident in the abdomen cavity, and in the uterus and vagina a brown mass was found. No treatment-related gross pathology findings were reported in any animals.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Hyperaemia was noted in liver and kidneys in a majority of control and high-dose males, and in liver, kidneys and ovary in some control and high-dose females. This is due to the method of sacrifice, which can cause blood to collect in major organs. In the mid-dose female that died during delivery, hemosiderosis in the right kidney and inflammatory cells in the liver were evident. Of the two high-dose females that died during the study period, purulent endometritis and catarrhal bronchitis was reported in one, while the other had lung emphysema. No treatment-related histopathological effects were reported in any animals.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were observed up to and including the highest dose level

2

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were observed up to and including the highest dose level

3

Dose descriptor:

NOAEL

Effect level:

>= 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were observed up to and including the highest dose level

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
There was no statistically significant difference in live birth index and viability index between the control group and treament groups (see Table 3). A slight reduction in the viability observed on lactation day 1 in the mid- and high-dose pups falls within the expected range, as shown with historical data (see Table 4), and is therefore not related to treatment with the test substance.

CLINICAL SIGNS (OFFSPRING)
No clinical signs were observed.

BODY WEIGHT (OFFSPRING)
A slightly lower body weight was reported in the high-dose pups at birth and on lactation day 4, although the difference was not statistically significant. Furthermore, the slightly lower mean weight of mothers from the start of the study throughout the gestation period is likely to have affected the birth weight of the pups. This effect is therefore not considered to be substance-related.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

>= 750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were observed up to and including the highest dose level

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Food consumption, females, gestation period

		Mean food consumption (g)/week
Dose (mg/kg bw/day)	N	1	2	3
Control	11	88.12 ± 22.72	142.27 ± 19.41	160.46 ± 29.02
30	11	96.36 ± 21.92	133.64 ± 11.85	171.82 ± 37.90
150	11	87.73 ± 15.55	124.09 ± 13.93*	171.82 ± 18.88
750	12	87.50 ± 20.17	120.83 ± 10.84*	164.17 ± 46.65

* p≤ 0.05

 

Table 2: Food consumption, females, lactation period

	Mean food consumption (g)
Dose (mg/kg bw/day)	N	Day 1-4	Per day
Control	8	61.25 ± 22.16	15.38 ± 5.59
30	7	64.29 ± 22.63	16.07 ± 5.66
150	6	76.67 ± 11.26	19.17 ± 2.81
750	7	51.43 ± 17.25	12.86 ± 4.31

Table 1: reproduction and developmental parameters

Observations	Dose (mg/kg bw/day)
	Control	30	150	750
Pairs (N)	12	12	12	12
Females with evidence of copulation (N)	10	12	11	12
Females achieving pregnancy (N)	11	11	11	12
Conceiving days 1-5 (N)	10	11	11	11
Conceiving days 6-12 (N)	1	0	0	0
Pregnancy ≤ 21 days (N)	0	0	0	0
Pregnancy = 22 days (N)	8	6	4	1
Pregnancy ≥ 23 days (N)	2	5	4*	11
Corpora lutea/ dam (mean)	13.33 ± 4.34	13.82 ± 2.09	14.00 ± 1.41	13.42 ± 1.24
Implants/ dam (mean)	13.18 ± 2.56	12.73 ± 1.90	12.00 ± 3.13	11.67 ± 2.23
Dams with live pups at birth (N)	10	10	7	9
Total number of live/dead pups at birth (N)	104/30	106/26	64/39	60/45
Dams with live pups on day 4 post-partum (N)	8	7	6	7
Total number of live pups on day 4 post-partum (N)	97	83	56	55
Live pups/ dam at birth (mean)	10.40 ± 4.38	10.60 ± 3.47	9.14 ± 4.02	6.67 ± 3.54
Live pups/ dam on day 4 post-partum (mean)	12.13 ± 1.89	11.86 ± 3.39	9.33 ± 4.37	7.86 ± 2.91
Sex ratio (m/f) on day 4 post-partum	0.4/0.6	0.4/0.6	0.4/0.6	0.5/0.5
Litter weight at birth (mean)	59.00 ± 21.83	60.00 ± 17.00	52.14 ± 20.79	41.67 ± 22.64
Litter weight on day 4 post-partum (mean)	81.25 ± 7.91	82.86 ± 9.94	70.83 ± 22.68	67.86 ± 20.99
Pup weight at birth (mean)	6.16 ± 1.46	5.78 ± 0.80	5.95 ± 1.31	6.18 ± 0.85
Pup weight on day 4 post-partum (mean)	6.83 ± 1.10	7.45 ± 1.91	8.43 ± 2.36	9.17 ± 1.82
Abnormal pups
Dams with 0	0	0	0	0
Dams with 1	0	0	0	0
Dams ≥ 2	0	0	0	0
Loss of offspring
Pre-implantation (corpora lutea minus implantation)
Dams with 0	6	4	6	6
Dams with 1	2	4	0	1
Dams with 2	2	4	1	0
Dams with ≥ 3	2	0	4	5
Pre-natal / post-implantations (implantation minus live birth)
Dams with 0	5	5	2	0
Dams with 1	2	1	1	1
Dams with 2	0	1	1	4
Dams with ≥ 3	5	5	5**	6
Post-natal (live births minus alive on lactation day 4)
Dams with 0	9	9	9	9
Dams with 1	2	0	0	1
Dams with 2	0	0	0	0
Dams with ≥ 3	1	3	1**	1***

* For 1/4 dams the birth was not observed, 1/4 dams died before the birth, 1/4 dams died during the birth

** 1 dam died before birth, 1 dam died during the birth, these are not counted

*** 1 dam died before birth, and is not counted

Table 4: Historical data from control groups treated with olive oil (vehicle) only

Parameter	Range
Live pups/dam at birth	3-15
Pup survival to day 4	3-15