Reaction products of 2,2-dimethylpropane-1,3-diol with 1-choloro-2,3-epoxypropane

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / 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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016 July 29

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Synonym: Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane
CAS number: 17557-23-2

Species:

rat

Strain:

Wistar

Remarks:

Rat, Han:WIST of Wistar origin

Details on species / strain selection:

Species / Strain: Rat, Han:WIST of Wistar origin
Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
Number of animals: 48 males, 48 females (nulliparous, non-pregnant females)
Number of animals/group 12 animals/sex in the control and dose groups (at least 8 pregnant female animals per group were expected)
Number of animals ordered: 55 male and 65 female animals; Randomization reserve was excluded from the study just after randomization.
Age of animals at start of Male animals: 83 – 85 days
the treatment: Female animals: 83 – 85 days
Body weights at start of 345 – 408 g for male animals
the study: 198 – 248 g for female animals
The weight variation did not exceed  20 per cent of the mean weight.
Animal health: Only healthy animals were used for the study. Healthy status was certified by the breeder (Appendix 19).
Acclimatization time: 20 days

Reason for selection of species

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal husbandry

Animal room no.: 24
Housing: Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Mated females: individually
Males after mating: 2 animals / cage
Cage type: Type III polypropylene/polycarbonate;
Bedding: Certified laboratory wood bedding (SAFE 3/4-S-FASERN Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-73494 Rosenberg Holzmühle 1 Germany see Appendix 22).
The bedding is suitable as nesting material. Details of quality of bedding material were reported.
The cages and bedding were changed once or twice a week.
Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: Above 10 air-exchanges/ hour by a central air-condition system.
Environmental conditions were maintained by an air-conditioning system. Temperature and relative humidity were verified and recorded daily during the study.

Route of administration:

oral: gavage

Vehicle:

other: sunfloweer oil

Details on exposure:

Table 3: Experimental design
Groups Dose
(mg/kg bw day) Dose volume
(mL/kg bw) Number of animals
Male Female
Group 1 0 5 12 12
Group 2 80 5 12 12
Group 3 245 5 12 12
Group 4 750 5 12 12
Animals in Group 1 only received the vehicle.

Details on mating procedure:

Mating begun 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) placed in a single cage. Females remained with the same male until copulation occurred.
Two pairs (animals no. 409-429 and animals no. 411-431, male-female, respectively) at 750 mg/kg bw/day failed to mate during 14-day although, these female animals showed regular cycle during the mating period. Therefore, the mating period was prolonged and these pairs were changed between each other from mating day 15 until successful mating on 17th or 19th day of mating.
Vaginal smears were examined for the presence of vaginal plug or sperm. Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually. Mating pairs were clearly identified in the raw data.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study.
Three aliquots of 10 mL of each formulation and three aliquots of control substance (vehicle) were taken two times and were analyzed.
Date of sampling: June 20 and July 25, 2022
Date of analysis: June 21 and July 26, 2022

Concentration of the test item in the dosing formulations varied between the range of 97 % and 107 % in comparison to the nominal values. Results and details of analysis are attached to the Report (Appendix 18.2).
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front in a GLP compliant study.
The recovery of the test item from the vehicle was within the acceptance criteria at ca. 1 mg/mL and at ca. 200 mg/mL.
Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane proved to be stable in sunflower oil at the intended concentrations at room temperature for six days.
A separate analytical report (Study no. 555-100-6268) provided these data

Duration of treatment / exposure:

The experimental period involved 20 days of acclimatization (including 14 days for examination of estrous cycle) and 50 (male), 43-63 (female) days treatment/ observation period (depending on the effectiveness of mating) and necropsy days.
The day of first treatment is considered as Day 0 of examination.

Frequency of treatment:

daily

Details on study schedule:

Study design

Dosing of both sexes begun after 20 days acclimatization – including 14 days pre-treatment estrous cycle examination – and was continued up to and including the day before the necropsy. Rats of this strain reach full sexual maturity at the age of 10 weeks. The mating phase started after 14-days treatment (pre-mating) period.
The test item was administered in a single dose by oral gavage on a 7 days/week basis, every day at a similar time (±2 hours). Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Male animals were dosed for 50 days (14 days pre-mating and 1-20 days mating, plus
16-35 days of post-mating period; until the necessary number of pregnant female animals was evident); then they were sacrificed on Day 50.
Females were dosed for 14 days pre-mating, through 1-19 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice on Days 43-63.
The day of birth (viz. when parturition is complete) was defined as day 0 post-partum.
Control animals were handled in an identical manner to the test groups receiving vehicle (5 mL/kg bw).
All animals were subjected to a full detailed gross necropsy. The brain, testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole were weighed in all male animals.
Five dams and males these females cohabited with were randomly selected from control, low and mid dose groups for additional examinations: functional observation battery (FOB), clinical pathology examinations, organ weighing and for a full histopathological investigation. There was no fertile pairing at 750 mg/kg bw/day, therefore five male animals in the high dose group were also randomly selected and examined for these endpoints. For five selected female animals at 750 mg/kg bw/day, clinical pathology examinations and organ weights were excluded from evaluation because these were non-pregnant and were subjected to necropsy at an earlier time.
F1 offspring were observed for clinical signs, litter weight, body weight, anogenital distance and nipple retention. Blood samples for serum FT3, FT4 and TSH assessment were pooled from 3-7 pups per litter on post-natal day 4 (in litters with at least 10 pups) and from 1-8 pups per litter on post-natal day 13. Remaining pups were euthanized on post-natal day 13 or shortly thereafter.

Dose / conc.:

750 mg/kg bw/day

Dose / conc.:

245 mg/kg bw/day

Dose / conc.:

80 mg/kg bw/day

Dose / conc.:

0 mg/kg bw/day

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Positive control:

no

Parental animals: Observations and examinations:

Examination of placental sign

All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestation day 13. If the test was negative on day 13, the examination was repeated on day 14 of gestation. Examination of placental signs was only for checking the pregnancy and data were not reported.

Observation of the delivery process

Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible from gestational day 21 onwards. All observations and any evidence of abnormal deliveries were considered and recorded. The duration of gestation was recorded and was calculated from day 0 of pregnancy.

Oestrous cyclicity (parental animals):

Estrous cycle

Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization.
Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation.
Vaginal smears were prepared and evaluated in each female animal on the day of necropsy.
Vaginal smears were stained with 1 % aqueous methylene blue solution then were examined with a light microscope.

Sperm parameters (parental animals):

Detailed histological examination was performed on the ovaries, uterus with cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Litter observations:

Observation of the offspring

Each litter were examined as soon as possible after delivery (within 24 h of parturition), to establish the number and gender of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.
Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the day after parturition was complete, post-natal day 0), and on post-natal day 13.
Litter weight were determined with an accuracy of 0.1 g. The litter weight was calculated by the individual weight of pups on post-natal day 4. Any abnormal behavior of the offspring was recorded.
All litters were checked and recorded daily for the number of viable and dead pups.
The anogenital distance of each pup was determined on post-natal day 4. The anogenital distance was normalized to the cube root of body weight. Therefore, individual body weight of pups was also determined with an accuracy of 0.01 g on post-natal day 4.
The number of nipples/areolae in male pups was counted on post-natal day 13.
Dead or stillborn offspring were subjected to necropsy by macroscopic examination on the day when they were found dead. On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test).

Postmortem examinations (parental animals):

Clinical pathology

Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group – except for high dose females – one day after the last treatment (i.e., on the day of necropsy).
Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran CP® anesthesia.
Three samples were taken from each animal: one for hematology, one for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry and for determination of serum levels of thyroid hormones (FT3, FT4 and TSH).

Gross necropsy was performed on each animal.
- Parental male animals: on Day 50.
- Dams not selected for toxicology examinations: on post-partum day 14 or shortly thereafter (Days 50, 52, 55 or 63);
- Dams selected for toxicology examinations: shortly after post-partum day 13 (post-partum days 14-16) on Day 52.
- Not delivered pregnant and non-pregnant female animals: on Day 43, 44, 55 or 56
- Offspring: on post-natal days 14-16.
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded

Organ weight

At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. Paired organs were weighed together.
In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.
Absolute organ weight was recorded. Relative organ weights (to body and brain weight) were calculated and reported.
Organ weights of randomly selected non-pregnant female animals at 750 mg/kg bw/day were determined and reported but were not evaluated.

Histopathology

Detailed histological examination was performed on the ovaries, uterus with cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (750 mg/kg bw/day) groups.
Based on macroscopic findings in high dose treated animals, the stomach was preserved and histologically processed and evaluated in all animals (male and female, control, 80, 245 and 750 mg/kg bw/day).
Additionally, kidneys with necropsy findings, sexual organs of not delivered pregnant female animal and its mating partner were processed and evaluated histologically as follows:
- Kidneys: male animals no. 108, 202, 208, 212, 306
- female animals no. 323, 326
- Prostate, seminal vesicle, testes, epididymides: male animal no. 307
- Ovaries, uterus, cervix, vagina: female animal no. 327
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by a light microscopy.

Postmortem examinations (offspring):

Gross necropsy was performed on each animal.
- Offspring: on post-natal days 14-16.
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded

Statistics:

Statistical evaluation

The statistical evaluation of appropriate data (marked †above) was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.
Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Reproductive indices:

Formulas for calculation of mating and fertility indices

Copulatory index: Measure of animals’ ability to mate
Males ("Number"  "of"  "males"  "with"  "confirmed"  "mating" )/"Total Number of males cohabited"  x "100"
Females ("Number"  "of"  "sperm positive"  "females" )/"Total Number of females cohabited"  x 100

Fertility index: Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.
Males ("Number"  "of"  "males"  "impregnating"  a "female" )/"Total Number of males with confirmed mating"  x 100
Females ("Number"  "of"  "pregnant"  "females" )/"Number of sperm positive females"  x 100

Gestation index: Measure of pregnancy that provides at least one live pup
("Number"  "of"  "females with live born pups" )/"Number of pregnant females"  x "100"

Offspring viability indices:

Formulas for calculation of pup mortality and sex ratio indices

Post-implantation mortality (intrauterine mortality)
("Number"  "of"  "implantations - Number of liveborns" )/"Number of implantations"  x 100

Post-natal mortality
("Number"  "of"  "liveborns -Number of live pups on PN13" )/"Number of liveborns"  x 100

Survival Index
("Number"  "of"  "live"  "pups"  "on postnatal day 13" )/"Number of pups born"  x 100

Sex ratio
("Number"  "of"  "pups"  "examined"  - "Number"  "of"  "males " ("females" ))/"Number of pups examined"  x 100

Normalized anogenital distance
"Absolute anogenital distance" /"(Body weight)1/3"

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

effects observed, treatment-related

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 245 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: na

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 750 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

other: na

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 245 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: na

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 245 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Remarks on result:

other: na

Critical effects observed:

no

Lowest effective dose / conc.:

750 mg/kg bw/day (nominal)

System:

other: not applicable

Organ:

other: not applicable

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Under the conditions of the present study, Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane caused clinical signs (male and female), depressed body weight development and reduced food consumption (male), changes in white blood cell parameters, macroscopic and histopathology changes in the stomach (male and female) in Han:WIST rats at 750 mg/kg bw/day by oral gavage as investigated in this study.
80 and 245 mg/kg bw/day did not cause local or systemic effects in parental male and female Han:WIST rats.
750 mg/kg bw/day severely impaired the impregnation in parental animals.
At 80 and 245 mg/kg bw/day, the reproductive performance (gonad function, mating behavior, conception, parturition) was unaffected.
Local effect was detected macroscopically and microscopically in the forestomach (squamous cell hyperplasia and ulceration) in male and female animals at 750 mg/kg bw/day. This local gastric effect induced clinical signs, effects on body weight, food consumption, changes in white blood cell parameters mainly in male animals.
The development of the F1 offspring was not impaired from birth up to post-natal day 13 as far as investigated in this study after repeated oral administration of dams at 80 and 245 mg/kg bw/day.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) and No Observed Adverse Effective Concentration (NOAEC) were determined as follows:
NOAEL for systemic toxicity of male rats: 245 mg/kg bw/day
NOAEL for systemic toxicity of female rats: 750 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats: 245 mg/kg bw/day
NOAEL for F1 Offspring: 245 mg/kg bw/day
NOAEC (local effects stomach): 49 mg/mL

Executive summary:

The purpose of this study was to obtain initial information on the systemically toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 80, 245 and 750 mg/kg bw/day compared to control animals according to OECD 422.

As a screening test, it was intended to provide initial information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses.

Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane was administered orally (by gavage) once daily at 0 (vehicle only), 80, 245 and 750 mg/kg body weight/day (mg/kg bw/day) doses to four groups of Han:WIST rats consisting of 12 animals per sex per groups in concentrations of 16, 49 and 150 mg/mL corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (sunflower oil) treated animals served as a control.

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane was stable in the vehicle in concentrations of 1 mg/mL and 200 mg/mL at room temperature for six days.

The concentration of the test item in the dosing formulations administered to the animals was checked twice during the study. Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane concentrations in the dosing formulations varied within the range of 97 % and 107 % (in comparison to the nominal values) and confirmed the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 50 days). Dams were additionally exposed through the gestation period and up to lactation days 13-15, i.e., up to the day before necropsy (altogether for
50-63 days, depending on mating days). Not delivered pregnant and non-pregnant female animals were administered for 43-56 days.

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period and during the mating period until evidence of copulation. Vaginal smears were also prepared and investigated on the day of the necropsy for each female animal.

The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 3-7 pups per litter (in litters with at least 10 pups) on post-natal day (PND) 4, from all dams and from 1-8 pups per litter on post‑partum/post-natal day 13 and from all parent male animals at termination. Serum level of FT3, FT4 and TSH were determined in the samples of parental male animals and PND 13 offspring.

Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology and blood coagulation, clinical chemistry, gross necropsy, organ weight and histopathology examination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.

Thyroid glands were preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination.

Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals (male and female) in the control and high dose groups.

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. In addition, the stomach was processed and evaluated histologically in all male and female animals in control, low, mid and high dose groups based on necropsy findings. Kidneys showing macroscopic findings at the necropsy were also processed and examined histologically in parental animals and in the low and mid dose groups.

 

The results of this study were summarized as follows:

Mortality

There was no mortality in the control, 80, 245 or 750 mg/kg bw/day groups during the course of study (male and female).

Clinical and functional observation

Test item related clinical signs were detected at 245 mg/kg bw/day (salivation, male) and at 750 mg/kg bw/day (decreased activity (male), salivation, enhanced urination, male and female) at the daily clinical observations.

Salivation and decreased activity were likely linked to the irritation noted in the stomach of affected animals at 750 mg/kg bw/day. Enhanced urination (wet bedding material in cages) referred to a diuretic effect of the test item at the 750 mg/kg bw/day dose.

All male and female animals exhibited normal behavior and physical condition with no abnormalities in the control, 80, 245 or 750 mg/kg bw/day groups at the detailed weekly clinical observations and at the functional observations.

Body weight and body weight gain

The body weight development was reduced in male animals at 750 mg/kg bw/day from Day 7 up to the termination of the treatment.

Food consumption

The mean daily food consumption was reduced – in accordance with the body weight depression – in male animals at 750 mg/kg bw/day during the pre-mating and post-mating periods.

A slight, toxicologically not relevant reduction in the mean daily food intake was transient in the male animals at 245 mg/kg bw/day and in the female animals at 750 mg/kg bw/day during the premating period.

Estrous cycle

A test item influence on the estrous cycle was not detected at any dose level (80, 245 or 750 mg/kg bw/day).

Delivery and pregnancy data of female animals

The pregnancy and delivery data were severely impaired in the female animals at 750 mg/kg bw/day. There were no implantation sites (macroscopic sign of pregnancy) in any of the female animals at 750 mg/kg bw/day

Reproductive performance

The reproductive performance was severely impaired at 750 mg/kg bw/day. The fertility index was 0 % at 750 mg/kg bw/day notwithstanding the copulatory index was 100 % both for male and female animals in the high dose group.

Clinical pathology – Hematology, blood coagulation, clinical chemistry

Clinical pathology investigation did not reveal test item related adverse changes in the examined hematology, blood coagulation or clinical chemistry parameters at 80, 245 or 750 mg/kg bw/day.

Slight changes in the white blood cell parameters – WBC, NEU and LYM – in the male animals at 750 mg/kg bw/day were presumably in accordance with gastric lesions detected at the necropsy and histopathology examinations.

Serum thyroid hormones

The thyroid hormone (FT3, FT4 and TSH) levels were not affected by the test item in parental male animals and in offspring sampled on post-natal day 13.

Necropsy

Gross necropsy observations revealed test item related changes in the stomach (thickening of the mucous membrane at cardia) at 750 mg/kg bw/day (male and female), which was due to the local effect of the test item and was verified histologically.

Organ weight

There were no adverse test item related alterations in the weights of the examined organs in male or female animals at 80, 245 or 750 mg/kg bw/day.

Histopathology

The investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands, ovaries, uterus, cervix, vagina) were histologically normal and characteristic on the sexually mature organism in the male and female animals in control and 750 mg/kg bw/day groups.

Histological examination revealed squamous cell hyperplasia in the non-glandular area of stomach, accompanied in some cases with ulceration in male and female animals at 750 mg/kg bw/day. Squamous cell hyperplasia is indicative of a restorative reaction following the irritative effect of the test item on the mucous membrane

 

 

Offspring

The offspring’s development was undisturbed at 80 and 245 mg/kg bw/day from birth to post-natal day 13. No effect on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

 

 

Conclusion

 

Under the conditions of the present study, Reaction products of 2,2-dimethylpropane-1,3-diol with 1-chloro-2,3-epoxypropane caused clinical signs (male and female), depressed body weight development and reduced food consumption (male), changes in white blood cell parameters, macroscopic and histopathology changes in the stomach (male and female) in Han:WIST rats at 750 mg/kg bw/day by oral gavage as investigated in this study.

80 and 245 mg/kg bw/day did not cause local or systemic effects in parental male and female Han:WIST rats.

750 mg/kg bw/day severely impaired the impregnation in parental animals.

At 80 and 245 mg/kg bw/day, the reproductive performance (gonad function, mating behavior, conception, parturition) was unaffected.

Local effect was detected macroscopically and microscopically in the forestomach (squamous cell hyperplasia and ulceration) in male and female animals at 750 mg/kg bw/day. This local gastric effect induced clinical signs, effects on body weight, food consumption, changes in white blood cell parameters mainly in male animals.

The development of the F1 offspring was not impaired from birth up to post-natal day 13 as far as investigated in this study after repeated oral administration of dams at 80 and 245 mg/kg bw/day.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) and No Observed Adverse Effective Concentration (NOAEC) were determined as follows:

NOAEL for systemic toxicity of male rats:                        245 mg/kg bw/day

NOAEL for systemic toxicity of female rats:                    750 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats:                   245 mg/kg bw/day

NOAEL for F1 Offspring:                                                   245 mg/kg bw/day

NOAEC (local effects stomach):                                                      49 mg/mL

 

 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

245 mg/kg bw/day

Study duration:

subchronic

Species:

rat