Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral (OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, rat): NOAEL (reproduction) ≥ 1000 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May - 22 Sept 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Government of India, Department of Science and Technology, National Good Laboratory Practice (GLP) Compliance Monitoring Authority (NGCMA)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
recommended by regulatory agencies
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Biosciences, Inc., USA, through its representative Vivo Bio Tech Ltd., Telangana, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation:males: 303 - 389 g (mean 356.30 g) / females: 210 - 242 g (mean: 226.33 g)
- Housing: Maximum three animals per sex and cage in solid bottom polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilized corn cob.
- Diet (ad libitum): 'Altromin' brand extruded pelleted rat feed manufactured by Altromin Spezialfutter GmbH & Co. KG, Germany
- Water (ad libitum): Potable water, passed through 'Aquaguard' water filter, and subjected to ultra violet irradiation
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: daily
Dosing formulations were prepared freshly each day before dosing. Corn oil was used as vehicle control item. The test item was completely miscible in corn oil. Required amount of test item was weighed on an analytical balance, then corn oil was added gradually to achieve appropriate concentrations i.e. 20, 60 and 200 mg/mL to meet dosage level requirements.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no.: 040516
- Date of Manufacture: 4 May 2016
- Date of Expiry: 3 May 2018
- Storage Conditions: room temperature (27 +/- 9 °C)
- Manufacturer: Medcraft International Pvt. Ltd. New Delhi, India
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: evidence of sperm in the vaginal smear, referred to as Day 0 of pregnancy

In case pairing was unsuccessful, re-mating of females with proven males of the same group was considered. Females showing no evidence of copulation were sacrificed 25 days after the last day of the mating period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item in the vehicle for the highest and lowest concentrations were determined at the Analytical Chemistry Section of the test facility in a separate study. A validated HPLC-UV method was used for identification and quantification of the test item in the vehicle. The HPLC method has been developed in the test facility. Test item formulations were subjected to verification for highest and lowest concentrations twice (i.e. in first week after initiation of treatment and in last week at termination of treatment) during the study.
Duration of treatment / exposure:
Males: minimum four weeks (minimum of two weeks prior to mating, during the mating period and, approximately, two weeks post-mating).
Females: throughout the study (two weeks prior to mating, variable time to conception, during pregnancy and thirteen days after delivery).

Dosing was continued in both sexes during the mating period. Males were further dosed after the mating period until the minimum total dosing period of 28 days had been completed. They were then sacrificed. Daily dosing of the parental females were continued throughout pregnancy and up to, and including, day 13 post-partum (the day before sacrifice).
Frequency of treatment:
7 days/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
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:
Males: 10
Females: 15
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day dose range finding study
Parental animals: Observations and examinations:
Mating date, confirmation of pregnancy, and delivery day were recorded. The duration of gestation was recorded and was calculated from day 0 of pregnancy. Dams were observed for signs suggestive of abortion or for premature delivery.

DETAILED CLINICAL OBSERVATIONS: Yes:

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND: Yes

for further details see Section 7.5.1
Oestrous cyclicity (parental animals):
A total of 60 females was screened for normal oestrous cycle for 2 weeks prior to the treatment period. All females exhibited an oestrous cycle of 4-5 days. Stages of the oestrous cycle were monitored daily in the females randomly assigned to four groups (15 females/group), from the beginning of the treatment period until evidence of mating.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: The testes, epididymides, prostate, seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernosus muscle complex, Cowper's glands and glans penis of all male adult animals were weighed on termination of treatment.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups/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, anogenital distance (AGD), presence of nipples/areolae in male pups, Estimation of Thyroid Hormones (Total T4)
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals which delivered: Day 14 post-partum
- Maternal animals which failed to deliver: 25 days after the last day of the mating period

GROSS NECROPSY
- Gross necropsy consisted of: see details under 7.5.1
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
see details under 7.5.1
Postmortem examinations (offspring):
SACRIFICE
- Pups were sacrificed on day 13 post-partum.
- Dead pups and pups killed on day 13 post-partum were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Blood samples from the day 13 pups were assessed for serum levels of thyroid hormone (T4).

GROSS NECROPSY
- Gross necropsy consisted of external examinations.
Statistics:
See Section 7.5.1
Reproductive indices:
For each group the following calculations were performed:
- Mating (%): Number of females mated/Number of females paired x 100
- Fertility index (%): Number of pregnant females/Number of females paired x 100
- Conception index (%): Number of pregnant females/Number of females mated x 100
- Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of post-natal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100
Offspring viability indices:
For each group the following calculations were performed:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of post-natal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100
Clinical signs:
no effects observed
Mortality:
no mortality observed
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:
effects observed, non-treatment-related
Description (incidence and severity):
Significantly low (p < 0.05) mean percentage PCV in high dose males and significantly high (p < 0.05) mean reticulocyte count percentage in high dose females were considered incidental because there was no dose-dependent change and the values were within respective historical control range.
Clinical biochemistry 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:
effects observed, non-treatment-related
Description (incidence and severity):
Some incidental and spontaneous lesions observed in animals from the high dose group (1000 mg/kg) comprised of bile duct hyperplasia in liver; ectopic thymus in thyroid and lymphocytic cell infiltration in prostate. All these changes were of minimal severity and were solitary in nature. These changes are commonly observed in rats and as they are known to be spontaneous in nature were not considered to be treatment related. Histopathological examination of the reproductive organs of male and female rats did not reveal any treatment related morphological alterations.
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
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenic cycle did not provide any evidence of impaired spermatogenesis, as explored by histological examination of testes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Histological examination of the uterus epithelium and endometrial glands did not reveal any treatment-related influences on oestrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment-related effects on reproductive parameters were noted. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment-related effects observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

For further details see 7.5.1
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Embryotoxic / teratogenic effects: no effects observed

MORTALITY AND CLINICAL SIGNS:
Treatment of dams with the test item did not have any adverse effect on the survival of the offspring during the lactation period. The incidence of litters with still-born pups, pups found dead in cage or cannibalized pups was very small and/or comparable across the treated and the control groups. Treatment did not induce any abnormal clinical signs, indicative of systemic toxicity, in offspring during lactation period.

BODY WEIGHT:
The mean litter body weights and mean litter body weight gain did not differ significantly (p > 0.05) from those of the concurrent control group during the post-natal lactation period except in high dose group pups. However, on day 2 of the post-natal lactation period, there was a significantly reduced (p < 0.05) body weight gain. Since the same group pups had gained weight at all other time points up to 13 days which was comparable to that of the control animals at all time points, this finding is considered to be of no toxicological significance.

ANOGENITAL DISTANCE:
No adverse effect on the anogenital distance for male and female pups was observed as the average of the anogenital distance (mm) was found to be comparable to that of control male and female pups from the post-natal day (PND) 0 to PND 4.

NIPPLE RETENTION:
The test item did not influence the nipple retention in male pups at post-natal day 12.

CLINICAL CHEMISTRY - Estimation of Thyroid Hormones (Total T4):
The test item did not induce any changes in the total T4 on day 4 after birth and also on day 13 of lactation.

ORGAN WEIGHTS:
Average absolute weights of thyroid glands from day 13 pups of all treatment group were found to be comparable with those of the control group.

EXTERNAL EXAMINATIONS:
Dead pups and pups killed on day 13 post-partum were carefully examined for external gross abnormalities. The total number of foetuses examined for external examination was 101 in the control group, 102, 100 and 120 in the 100, 300 and 1000 mg/kg bw/day dose groups, respectively. The incidence of normal foetuses and litters observed in this study was 100% in control group and in all treatment groups. There were no any external abnormalities observed in external genitalia or any other organs of pups.
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically relevant effects were observed up to and including the highest dose level
Reproductive effects observed:
no

Summary tables are provided in a pdf document attached to this IUCLID dossier.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance. The study is thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, 8.7, of Regulation (EC) No. 1907/2006 (REACH).
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 reproductive toxicity after oral exposure of reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and [2-[2-(2-butoxyethoxy)ethoxy]ethyl](3,6,9,12-tetraoxahexadecyl)adipate (EC No. 943-330-9) has been investigated in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 and under GLP conditions (Intox, 2017). Groups of 10 male and 15 female Wistar rats were administered with the test item daily by oral gavage at the doses of 100, 300 and 1000 mg/kg bw/day. A concurrent control group of 10 males and 15 females receiving the vehicle (corn oil) at 5 mL/kg bw was also maintained. Males were dosed for a period of 4 weeks, up to and including the day before scheduled sacrifice. Females were dosed for 50 – 60 days, throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy and thirteen days after delivery, up to and including the day before scheduled sacrifice. The rats were examined daily for signs of toxicity, morbidity and mortality. Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During pregnancy, females were weighed on days 0, 7, 14, 20 and then within 24 h of parturition (day 1 post-partum), at day 4 and day 13 post-partum. During pre-mating, food consumption was measured weekly. During pregnancy it was measured on gestation day 0, 7, 14 and 20 and during lactation on days 4 and 13. Vaginal smears of all females were monitored daily during pre-treatment, pre-mating treatment period and during mating period until evidence of mating. After parturition, each litter was examined for number and sex of pups, still births, live births, runts and the presence of gross abnormalities. Litters were weighed within 24 h of parturition (day 0 or 1 post-partum) and on day 4 and day 13 post-partum. The anogenital distance of each pup was measured daily on the post-natal day (PND) 0 to PND 4. Pup body weight was recorded daily on PND 0 to PND 4 and at termination on day 13. The numbers of nipples / areolae in male pups were counted on PND 12. Total T4 assessments were carried out on few pups on day 4 and day 13 after birth. All animals sacrificed terminally were subjected to a detailed necropsy and the weights of testes, epididymides, prostate, seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernous muscle complex, Cowper's glands, glans penis of all adult males, the ovaries and uterus with cervix of all adult females were recorded.

There was no incidence of any treatment-related mortality amongst the rats at any of the dose levels in both males and females. Treatment of males and females did not induce any adverse clinical signs at any dose levels. No abortion/premature deliveries were observed in any of the dose levels during the study period. No treatment-related gross pathological changes were noted. Histopathological examination was performed on tissues of the control and high dose group animals, where the changes in the high dose group were incidental or comparable to the control group or unrelated to treatment. The reproductive organ weight data, gross and histopathological examination of reproductive organs did not reveal any treatment-related changes. Data regarding development and reproduction indicated no difference between the animals treated with the test item and the vehicle control.

Based on the findings of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) of reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and [2-[2-(2-butoxyethoxy)ethoxy]ethyl](3,6,9,12-tetraoxahexadecyl)adipate (EC No. 943-330-9) in Wistar rats, following oral administration for a period of four weeks for males and 50 to 60 days for females for reproductive toxicity was found to be ≥ 1000 mg/kg bw/day, corresponding to the highest dose tested.

Effects on developmental toxicity

Description of key information

Oral (OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, rat): NOAEL (development) ≥ 1000 mg/kg bw/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:
The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance. The study is thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, 8.7, of Regulation (EC) No. 1907/2006 (REACH).
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

The reproductive toxicity after oral exposure of reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and [2-[2-(2-butoxyethoxy)ethoxy]ethyl](3,6,9,12-tetraoxahexadecyl)adipate (EC No. 943-330-9) has been investigated in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 and under GLP conditions. The study has been discussed in detail above. Here, only findings related to developmental toxicity are summarised.

No embryotoxic / teratogenic effects have been observed. Treatment of dams with the test item did not have any adverse effect on the survival of the offspring during the lactation period. The incidence of litters with still-born pups, pups found dead in cage or cannibalised pups were very small and/or comparable across the treated and the control groups. Treatment did not induce any abnormal clinical signs, indicative of systemic toxicity, in offspring during lactation period. The mean litter body weights and mean litter body weight gain did not differ significantly from those of the concurrent control group during the post-natal lactation period except in high dose group pups. However, on day 2 of the post-natal lactation period, there was a significantly reduced body weight gain. Since the same group pups had gained weight at all other time points up to 13 days which was comparable to that of the control animals at all time points, this finding is considered to be of no toxicological significance. No adverse effect on the anogenital distance for male and female pups was observed as the average of the anogenital distance was found to be comparable to that of control male and female pups from the post-natal day (PND) 0 to PND 4. The test item did not influence the nipple retention in male pups at post-natal day 12. It did not induce any changes in the total T4 on day 4 after birth and also on day 13 of lactation. The average absolute weights of thyroid glands from day 13 pups of all treatment groups were found to be comparable with those of the control group. Dead pups and pups killed on day 13 post-partum were carefully examined for external gross abnormalities. The total number of fetuses examined for external examination was 101 in the control group, 102, 100 and 120 in the 100, 300 and 1000 mg/kg bw/day dose groups, respectively. The incidence of normal fetuses and litters observed in this study was 100% in control group and in all treatment groups. There were no external abnormalities observed in external genitalia or any other organs of pups.

Based on the findings of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) of reaction mass of bis[2-[2-(2-butoxyethoxy)ethoxy]ethyl]adipate and [2-[2-(2-butoxyethoxy)ethoxy]ethyl](3,6,9,12-tetraoxahexadecyl)adipate (EC No. 943-330-9) in Wistar rats, following oral administration for a period of four weeks for males and 50 to 60 days for females for developmental toxicity was found to be ≥ 1000 mg/kg bw/day, corresponding to the highest dose tested.

Justification for classification or non-classification

The available data on toxicity to reproduction following exposure via the oral route do not meet the classification criteria according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.