Ethanol, 2,2'-oxybis-, 1,1'-diformate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

No studies are available on fertility based on no concern from repeated dose toxicity (90d oral gavage, rats, including some reproductive parameters) and pre-natal developmental toxicity studies.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

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 OECD 408 was performed following a deviation including detailed monitoring of parameters relevant for reproduction, in particular:

-weight of male and femal gonads and accessory sex organs

-stage of oestrous cycle and cycle duration for females

-histopathological examination of uterus and cervix

-histopathological examination, with special emphasis to stages of spermatogenesis, in males

-histopathology of interstitial testicular structure

Results:

Weight

There were no changes in terminal fasting body weights, absolute organ weights or organ to body/brain weight ratios which were related to treatment.

Gross Pathology

In treated rats, gross lesions involving uterus (dilatation) and testes (enlarged/small and flaccid) were associated with incidental microscopic findings

Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks prior to necropsy for main and recovery groups.

The calculated mean oestrous cycle length was 4.1, 4.3, 4.2 and 4.1 days in vehicle control, low, mid and high dose groups, respectively during treatment period.

During recovery period the mean oestrous cycle length was 4.3 and 4.4 days in vehicle control recovery and high dose recovery groups.

The mean oestrous cycle length for main and recovery groups was not significantly different from respective vehicle control groups.

The time interval (in days) from the diestrus of an estrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.

Histopathological examination

No effects on uterus, cervix, testes at micro level

Spermatogenesys (qualitative) :

no treatment related effects

Effects on developmental toxicity

Description of key information

OECD 414, rats, oral gavage, NOAEL maternal = 1000 mg/kg bw /day, NOAEL developmental = 1000 mg/kg bw /day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology (India) Pvt. Ltd. Plot 4B, MN Park, Shameerpet Mandal, Turkapally Village, Medchal District, Telangana -500078

-Age at the study initiation: 12 and 13 weeks

- Weight at study initiation: Mean body weight of Day 0 mated females
G1 : 241.99 ± 14.44
G2: 241.64 ± 11.95
G3: 242.38 ± 16.18
G4: 244.98 ± 15.63
The weight variation of rats was minimal and did not exceed ± 20 % of the mean body weight in each group.

- Housing: standard polysulfone rat cages (size: Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube. Additionally, polycarbonate rat huts were placed inside the cage as environmental enrichment objects which were replaced once a week.
i.Pre mating / Acclimatization: Two rats of the same sex per cage were housed.
ii. Mating: Female rats were cohabited with males in a 1:1 ratio in same cage.
iii. Post-mating / Treatment: After mating confirmation, females were housed individually. Nesting material (paper cuttings) were placed inside the cages from GD 18 onwards.

- Diet (e.g. ad libitum): ad libitum

- Water (e.g. ad libitum): ad libitum

- Acclimation period: After detailed clinical examination for good health and suitability for the study, 120 females and 50 male rats were acclimatized for five days before initiation of mating. Only nulliparous and non-pregnant females were used in the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22°C
- Humidity (%): 64-66%
- Air changes (per hr): 12 to 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12hrs dark/12 hrs light

IN-LIFE DATES: From: acclimatisation, mating period to 20 GD (females)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared fresh daily before dosing and used within the established stability period of 5 hours.
Based on the dose, required quantities of the test item was weighed in a beaker for each dose levels separately and small volume of vehicle [Milli-Q water] was added and stirred using a glass rod till a uniform solution was obtained. This was then transferred in to a measuring cylinder and the volume was made up to the required level using the vehicle to get the final desired concentrations of 10 (G2), 30 (G3) and 100 (G4) mg/mL.


VEHICLE
- Concentration in vehicle: from 0 to 100 mg/ml
- Amount of vehicle (if gavage): 10 ml (doe volume)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analysis of active ingredient (a.i.) were collected from the dose formulations intended for first treatment and last treatment (-4 days) for the first
batch of rats. The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as back up set which was
stored at ambient condition. For each set, composite samples were drawn from each dose formulation including vehicle control. The samples collected (both
the sets) were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the concentration of the dose formulation.
Dose formulation samples were analyzed for Diethylene Glycol Diformate content using analytical method which was validated
Dose formulations samples are considered acceptable if results are within ± 10.0 % of the claimed concentration and the relative standard deviation (% RSD), of assay is less than or equal to 5 %.
As the results from low dose from the first preparation was above the acceptance criteria, samples from subsequent preparation was used for analysis.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: until vaginal smear/vaginal plug exhibited sperm
the cohabitation continued until tehre was enough umber of pregnant females for the study
- Further matings after two unsuccessful attempts: [no / yes (explain)]: no
- Verification of same strain and source of both sexes: [yes / no (explain)]: no
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: both vaginal plug and vaginal smear refered as Day 0

Duration of treatment / exposure:

gestation day 5 to gestation day 19

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 for each dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a a preliminary dose range finding study (DRF) in pregnant rats carried out using 7 rats per group with Diethylene Glycol Diformate dosed at 100, 300 and 1000 mg/kg/day along with the concurrent vehicle control group. The rats were treated with the dose formulations by oral gavage at a dose volume of 10 mL/kg body weight from GD 5 to 19 and observed for clinical signs, mortality and bodyweight change and food consumption. The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live or dead fetuses. The number of corpora lutea was counted. All the fetuses were weighed, sexed, measured for anogenital distance, and examined for external malformations.
In this study, dose levels up to 1000 mg/kg/day were tolerated. No test item related clinical signs were noted at any dose. The body weight gains and food consumption were unaffected up to the highest dose of 1000 mg/kg/day. There was no fetal developmental toxicity up to the highest tested dose of 1000 mg/kg/day.

- stability of the dose solutions : The stability of Diethylene glycol diformate in vehicle was established at concentration of 0.1 and 150 mg/mL under validated method. The results indicated that the test item was stable and homogenous in the vehicle for 5 hours at both the concentrations when stored at room temperature.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During acclimatization period, all the rats were observed once daily.
Observations for clinical signs were performed twice a day, pre dose and post dose (30 minutes to 2 hour post dose, approx.) during treatment days and at least once on other days.
Each rat was observed twice daily during treatment period for morbidity and mortality i.e., once in the morning and once in the afternoon. Based on the assessment as there were no clinical signs of concern, the observation for morbidity and mortality was carried out once during weekends and public holidays

BODY WEIGHT: Yes
- Time schedule for examinations: All females included in the study were weighed on gestation days 0, 3, 5, 8, 11,
14, 17 and 20.

FOOD CONSUMPTION : Yes, About 200 g of food (food input) was provided on Day 0. The food output was recorded and replenished to about 200 g on Days 3, 5, 8, 11, 14 and 17 and food output on Day 20 of presumed gestation was recorded.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

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


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: gross pathological changes in placenta and in all visceral organs. Gross necropsy involved external observation and examination of thoracic and abdominal viscera including uterine contents. At necropsy, thyroid gland was collected and weighed from each rat (post fixation) and subjected to histopathological examination. The tissue was processed for routine paraffin embedding and 4-5 micron sections were stained with Mayer’s Haematoxylin and Eosin stain. Unused tissue will be archived.
List of analysed organs: eyes, bone, joints, femorotibial, gut associated lymphoid tissue, heart, ileum jejunum, kidneys, liver, lungs, mammary glands, lymph nodes, optic nerve, ovaries, oviducts, pancreas, pituitary gland, rectum, skin, sciatic nerve, salivary glands, spinal cord, spleen, sternum, stomach, thymus, thyroid and parathyroid, trachea, urinary bladder, uterus vagina.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: gross evaluation of placenta


The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes / No / No data
- Number of corpora lutea: Yes / No / No data
- Number of implantations: Yes / No / No data
- Number of early resorptions: Yes / No / No data
- Number of late resorptions: Yes / No / No data
- Other: gross placenta analysis

Uteri that appeared non-gravid were subjected to 10% ammonium sulphide staining to observe implantation sites if any (identified as pregnant rats) or to confirm the non-pregnant status.

Fetal examinations:

- External examinations: Yes:, all fetuses
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: Yes, half per litter

Moreover the following information were recorded:
Total number of fetuses
b. Number of live fetuses
c. Individual fetal body weight (g)
d. Anogenital distance (mm) from all live fetuses
e. Fetus sex -external determination based on anogenital distance that was confirmed based on gonadal examination (internal sex) during visceral examination.

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland was analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for
male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using
Kruskal Wallis test for group comparison. Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the group
differences were significant.

Indices:

The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s exact test for group association.
The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran Armitage trend test and pair wise comparison will be tested by Fisher’s exact test for group association.

Clinical signs:

no effects observed

Description (incidence and severity):

There was no morbidity or mortality and the treated rats were normal throughout the experimental period at all the doses tested.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The maternal absolute mean body weights were unaffected by treatment up to the highest dose of 1000 mg/kg/day. The absolute mean body weight gains were also comparable between vehicle control and treatment groups except for
occassional significant decrease during gestation days 8-11 (19% lower v/s vehicle control) and 17-20 (9% lower v/s vehicle control) were considered incidental due to the pattern of occurance, lack of dose dependency. Further the corrected body weight gain was also unaffected by treatment up to the highest dose of 1000 mg/kg/day, hence the decreases seen in body weight gains were considered incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The maternal mean food consumption was unaffected by treatment up to the highest dose of 1000 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological findings in any of the test item treated rats or vehicle control rats at necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Thyroid hormone levels (T3, T4 and TSH) were not affected across the treated groups when compared to the vehicle control group. There were no treatment-related changes in thyroid gland weights or microscopic alterations in all groups compared to the vehicle control group.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Details on maternal toxic effects:

No test item related changes were observed in maternal parameters (mean uterine weights, mean number of corpora lutea, implantations, early and late resorptions, pre-and post-implantation loss rates and rats with any resorption) at any dose tested. Gross evaluation of placenta revealed no findings

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

early or late resorptions

food consumption and compound intake

gross pathology

mortality

number of abortions

pre and post implantation loss

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The litter parameters (mean fetal weight and number of live fetuses) were comparable between the Vehicle control group and rats treated up to the highest dose of 1000 mg/kg/day

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

no dead fetusea were recorded

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The anogenital distance in male and female fetuses were comparable to the concurrent Vehicle control

External malformations:

no effects observed

Description (incidence and severity):

No test item-related changes were observed during external observations of fetuses of rats treated up to 1000 mg/kg/day.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no skeletal malformations observed in any litter at any of the tested dose levels except for one fetus from a dam (Rs9216) at 100 mg/kg/day which had absent caudal vertebrae, and 3rd and 4th sacral vertebrae which was
expected in a fetus which had thread like tail during external examination. Further similar observations were not observed at higher doses and hence the observed malformation was attributed to random occurence and not related to
treatment.
Few incidences of anomalies showed significant increase such as bilateral accessory rib No.14 at 300 mg/kg/day and right rudimentary rib No.14 at 1000 mg/kg/day as compared to vehicle control group. These incidences were
randomly distributed across the treatment and control groups and were not dose-related, consistent with historical controls and hence considered not related to treatment.

Visceral malformations:

no effects observed

Description (incidence and severity):

No test item-related changes were observed during fresh visceral examination of fetuses of rats treated up to 1000 mg/kg/day. However, incidences of variations / anomalies such as incidence of fetus with slight renal pelvis dilation at 1000 mg/kg/day (1/162 fetuses) and incidence of fetus with moderate renal pelvis dilation at 1000 (1/162 fetuses). These incidences were statistically comparable to vehicle control group. The incidence is attributed to random occurence that is consistent with historical controls.
Fetal heads in all dose groups were normal when subjected to Wilsons Razor blade sectioning

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

Developmental effects observed:

no

refer to the attached final report for detailed tables and further information

Conclusions:

The substance was tested pre-natal developmental toxicity to rats by oral route following OECD 414. Under the experimental conditions the NOAEL for maternal toicity was equal to 1000 mg/kg bw /day and for developmetal toxicity to 1000 mg/kg bw /day.

Executive summary:

The substance was tested for pre-natal developmental toxicity in rats buy oral route following OECD 4141. The objective of this study was to evaluate the developmental toxicity

(teratogenic) potential of the test item Diethylene Glycol Diformate to cause adverse effects on the pregnant female rats and development of the embryo and fetus consequent to exposure of Diethylene Glycol Diformate to pregnant rats by oral route during gestation days (GD) 5 to 19.

A total 96 of Day 0 pregnant rats1 were randomly divided into different groups according to the study design, and 24 pregant females were dosed at 0, 100, 300 and 1000 mg/kg bw /day.

The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival, number of corpora lutea, and fetus parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations]. Approximately half of the fetuses from

each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroids were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifie (GD20).

Results of the study are summarized below:

• Clinical signs and gross necropsy changes: There were no clinical signs, mortalities or gross pathological changes in treated rats at any of the doses tested.

• Maternal Parameters: No treatment-related effects on maternal body weights and food consumption up to the highest tested dose of 1000 mg/kg/day. The other maternal parameters comprising of uterine weight, number of corpora lutea, implantations and early and late resorptions, pre-and post implantation loss were comparable to vehicle control group up to the high dose of 1000 mg/kg/day. Gross evaluation of placenta revealed no findings.

• Litter Parameters: No treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights, anogenital distance in male and female fetuses, were observed.

• Fetal examination: The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day.

• Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with Diethylene Glycol Diformate up to the highest dose of 1000 mg/kg/day

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

• Maternal toxicity is 1000 mg/kg/day as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

• Fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score1 ) and consistent study performed following OECD 414 on the target substance diethylene glycole diformate

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Reproductive toxicity includes adverse effects on sexual function and fertility in sexually adult males and females animals, as well as developmental toxicity in the offspring. However, developmental toxicity essentially means all the adverse effects induced during pregnancy that can be manifested at any point of the life span of the animal, which might in turn bring to structural abnormality, altered growth and/or organs development, functional deficiency, even death.

Table 3.7.1(a) of Annex I of EC Regulation 1272/2008 states that to classify compounds "for category 2 suspected human reproductive toxicant, reproductive effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects". To this extent the pre-natal developmental toxicity study OECD 414 and the repeated dose toxicity study OECD 408 enhanced with some reproductive parameters do not provide any indication of direct adverse effect on reproduction and/or development. In fact up to the dose of 1000 mg/kg bw/day no effects were observed in the sex organs weights, histopahtology, oestrus cycle and spermatogenesys. Moreover, no developmental toxic effects in the offspring were observed at all doses.

In conclusion, since no adverse effects on reproduction were observed, classification for reproductive/developmental toxicity is not warranted under Regulation 1272/2008