Reaction mass of 5,8,11,14-Tetraoxaoctadecane and 5,8,11,14,17-Pentaoxaheneicosane

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:

2011-11-09 to 2013-05-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Health Effects guidelines, OPPTS 870.3650, Combined repeated dose Toxicity study with the Reproduction/ developmental Toxicity
screening Test. EPA 712-C-00-368, July 2000.
Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5,8,11,14,17-pentaoxahenicosane; 5,8,11,14-tetraoxaoctadecane

EC Number:

940-417-3

Molecular formula:

C14H30O4 C16H34O5

IUPAC Name:

5,8,11,14,17-pentaoxahenicosane; 5,8,11,14-tetraoxaoctadecane

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

CAS No.: 31885-97-9
Chemical Name: Poly(oxy-1,2-ethanediyl),a-butyl-w-butoxy-
pH: 6-7 (20 oC, 100 g/l)
Physical State at RT: liquid
Density: 0.92 g/cm3 (20°C)
Colour: colourless
Storage Conditions: at room temperature
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 9-10 weeks old, females: 8-9 weeks old.
Body weight at the allocation of the animals to the experimental groups: Females: 171 to 202 g, (mean: 186.50 g, ± 20%= 37.30 g)
Males: 237 to 285 g, (mean: 264.21 g, ± 20%= 52.84 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3°C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male),
type III H, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made.
Before the first administration all animals used for the study were weighed and assigned to the experimental groups
with achieving a most homogenous variation in body weight throughout the groups of males and females.

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

The test item was weighed into a tared plastic vial on a suitable precision balance and the sterile water was added to give the appropriate
final concentration of the test item based on the active ingredients. The formulation was placed on Vortex machine for short period to
ensure proper homogenistation of the formulation.
The vehicle has been selected as suggested by the sponsor and on the basis of the test item’s characteristics.
The test item formulation was prepared freshly on each administration day before the administration procedure.


The following doses were evaluated:
Control: 0 mg/kg body weight
Low Dose: 50 i.a mg/kg body weight
Medium Dose: 150 i.a mg/kg body weight
High Dose: 450 i.a mg/kg body weight

i.a = active ingredient

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The doses were selected on the basis of data from a Dose Range Finding Study.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle
using the same dose volume.

Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement are minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed for nominal concentration and homogeneity of the test item in the vehicle at various intervals.
Stability analysis was performed on samples collected from the top and bottom dose levels.
Samples for the nominal concentration verification was taken in study week 1 (first week of pre mating period), study week 3 (first week of mating),
study week 5 (gestation) and study week 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation in study week 1 and 5.
Samples for stability analysis was taken from top and bottom dose level in study week 1 at 0 hr and 6 hrs.
All concentration samples were stored frozen (approximately -20°C) until the analysis was performed.
The dose formulation analysis was performed at GLP-certified contract laboratory IBACON GmbH, Arheilger Weg 17, D-64380 Rossdorf,
Germany.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days,
i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and
up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Details on study schedule:

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible
after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum.
Live pups were identified by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 250, 750 mg/kg bw
Basis:
actual ingested

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and on lactation days in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.

FOOD CONSUMPTION:
- Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the
dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

OPHTHALMOSCOPIC EXAMINATION: yes

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
The males were sacrificed after the completion of the mating period (after a dosing period of 28 days) except half of the males in control and HD
groups, which were further kept for 14 days recovery post 28 days dose administration and then scheduled for necropsy. Females were subjected to necropsy on the respective post-natal day 4. Males and females were sacrificed using an anesthesia (e.g. ketamine/xylazin). The pups were killed by decapitation on post-natal day 4.
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Non-pregnant females (animal no. 54, 60 and 95) were sacrificed on study day 26 from the day of sperm-positive vaginal smear as an evidence
of mating.
All animals were subjected to a detailed gross necropsy, which includes careful examination of the external surface of the body, all orifices and
the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

The reproductive organs of all adult animals were weighed. The wet weight of the organs (liver, kidneys, adrenals, testes, epididymides, prostate,
seminal vesicles and coagulating glands, ovaries, uterus with cervix, thymus, thyroid/parathyroid glands, spleen, brain, pituitary gland, heart))
of all males of control and high dose groups (including the animals subjected to recovery), six randomly selected males of low and mid dose groups and six randomly selected females from each group were recorded as soon as possible. Paired organs were weighed separately.


The following tissues (brain (cerebrum, cerebellum and pons), ovaries (females), spinal cord, uterus with cervix (females),
liver, vagina (females), kidneys, testes (males), adrenal glands, epididymides (males), stomach, prostate and seminal vesicles
with coagulating glands as a whole (males), small and large intestines (including Peyer´s patches), urinary bladder,
thymus, lymphnodes (mesentric and axillary), Thyroid, peripheral nerve (e.g. sciatic nerve) with skeletal muscle,
spleen, bone with bone marrow (sternum), lung and trachea pituitary gland, mammary glands, oesophagus, heart, gross lesions)
of the same selected animals from each group were preserved in 10% neutral buffered formalin except eyes, testes and epididymides
that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 10% neutral buffered formalin.

HISTOPATHOLOGY: Yes

All organs and tissues listed were evaluated from all males of the control and high dose group assigned to main or recovery necropsy,
as well as from the following six randomly selected females from the control and high dose group (assigned to main necropsy):
Females Nos.: 51, 53, 55, 56, 58, 59, 85, 87, 89, 90, 92, 94.
In addition, liver, kidney, spleen and thyroid gland were also evaluated from the following six randomly selected males and females from the
low dose and medium dose groups: Males Nos.: 13, 15, 19, 21, 22, 24, 27, 28, 29, 32, 33, 36; Females Nos.: 63, 64, 66, 68, 71, 72, 73, 74, 75,
80, 81, 83.

Reproductive organs (ovary, uterus, cervix, vagina, testis, epididymis, prostate gland, seminal vesicle and coagulating gland) and
macroscopic changes were evaluated in all study animals.

For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of
additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd.
(test site for tissue processing), Willow Court, Netherwood Road, Hereford HR2 6JU, England. Histopathological evaluation was
performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology),
6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were
performed according to the corresponding SOP’s of the test sites.

Postmortem examinations (offspring):

Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

Statistics:

For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control- and test groups. The student-t test was used for the comparison of recovery control and recovery high dose groups.
These statistics were performed with GraphPad Prism V.5 software (p<0.05 was considered as statistical significant).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant toxic effect found up to the highest dose applied.

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:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant toxic effect found up to the highest dose applied

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

The data on the body weight development, hematology, clinical chemistry, organ weight and histopathology are given under 7.5.1

Table: Summary on the reproduction performance
	Dose
	Control	100 mg/kg bw	250 mg/kg bw	750 mg/kg bw
Pairs started	12	12	12	12
Females achieving pregnancy	10	12	12	11
Corpora lutea	13.70 ± 1.49	13.58 ± 1.88	14.50 ± 1.88	13.82 ± 3.16
Implantation sites	13.20 ± 1.32	11.58 ± 3.60	12.92 ± 1.51	11.82 ± 2.68
Dams with live young born	10	11	12	11
Live pubs born	11.90 ± 1.32	11.27 ± 2.65	11.50 ± 1.73	9.09 ± 4.61
Live pubs on postnatal day 4	11.20 ± 2.57	11.27 ± 2.65	11.42 ± 1.88	9.09 ± 4.61
Group mean litter weight at birth [g]	5.9 ± 0.5	6.0 ± 0.3	6.1 ± 0.5	6.1 ± 0.7
Group mean litter weight on postnatal day 4 [g]	9.9 ± 1.5	10.4 ± 1.0	10.0 ± 1.1	11.1 ± 4.4

Comments on the number of live pubs at birth: although no stastistical difference could be obtained for all dose groups, the value obtained for the high dose group is lower. The reduced value is due to three animals (one with four pubs, one with one pub, the other with two pubs). No biological significance could be attributed.

Applicant's summary and conclusion

Conclusions:

The reproduction toxicity of the registration substance was investigated according to the Guideline OECD 422. No indication of reproduction toxicity was found and the NOAEL of 750 mg/kg bw was obtained for the repeated dose toxicity and for the reproduction toxicity.

Executive summary:

The reproduction toxicity of the registration substance was investigated according to the OECD Gudieline 422. Twelve males and females each were treated at doses of 0, 100, 250 and 750 mg/kg bw via gavage.

The red blood cell was found as primary target in parental animals, whereas no adversity could be assigned.

Further, no significant effect on the reproduction performance as well as on the reproduction organs was found for all treated animals. The NOAEL of 750 mg/kg bw was obtained both for the repeated dose toxicity and the reproduction toxicity.