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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In a combined OECD 422/408 study no reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). Also a developmental neurotoxicity study (OECD 426) showed no adverse effects up to the highest dose level tested (1000 mg/kg).

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Details on species / strain selection:
Untreated, nulliparous, non-pregnant females and untreated males were used at the initiation of the study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- At initiation of dosing males and females were 6 weeks old, males weighed between 154 and 187 g and females weighed between 120 and 145 g.
- Fasting period before study: no
- Housing: Pretest and pre-mating: animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm); Mating: Males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm); Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm); Lactation: Females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) (During motor activity measurements, animals did not have access to food for a maximum of 2 hours)
- Water: Free access to tap-water (During motor activity measurements, animals did not have access to water for a maximum of 2 hours)
- Acclimation period: for 7 days prior to start of treatment;

DETAILS OF FOOD AND WATER QUALITY:
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
(set to maintain)
- Temperature (°C): 20 to 22
- Humidity (%): 43 to 66
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 April 2017 To: 02 August 2017
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily as solution (low and mid dose) or suspension (high dose) within 5 hours prior to dosing and were homogenized to a visually acceptable level. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.
Details on mating procedure:
After 8 weeks of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which the females who had not shown evidence of mating were separated from her male.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of samples of formulations taken in week 1, 5 and 13 during the treatment phase were analysed according to a validated method. The samples were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were in agreement with the target concentrations (i.e. 90-110% for the low and mid dose and 85-115% for high dose). Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of formulations over 5 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study.
Duration of treatment / exposure:
Males were treated for 92 days, i.e. 8 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were treated for 93-98 days, i.e. during 8 weeks prior to mating, the variable time to conception, the duration of the pregnancy and 14-15 days after delivery up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were treated for 85-96 days.
Routinely, females that are littering are left undisturbed. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
Frequency of treatment:
Once daily for 7 days per week
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:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Based on the results of a 14-day dose range finding study the dose levels for this combined 90-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg bw/day. In this DRF no mortality or no abnormalities were noted at 500 and 1000 mg/kg bw/day.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, 19 and 20 postcoitum and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION: Yes
- Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes were examined using an ophthalmoscope after application of a mydriatic agent prior to initiation of treatment in all animals, and towards the end of the treatment period in Week 13 in all males and during lactation in all females.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Blood of F1 was collected on PND 4 and PND 13-15 if possible.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes for F0, o/n (with a maximum of 24h)
- How many animals: all F0 animals; 2 pups per litter of F1
- Parameters checked according to Guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Blood of F1 was collected on PND 4 and PND 13-15 if possible.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes for F0,, o/n (with a maximum of 24h)
- How many animals: all F0 animals; 2 pups per litter of F1
- Parameters checked according to Guidelines. Included thyroid hormone analyzed (T4 and TSH).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations:
The selected males were tested during Week 13 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 8-12). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined:
5 animals/sex/group
- Battery of functions tested: hearing ability, pupillar reflex and static righting reflex, fore- and hind-limb grip strength, locomotor activity.

IMMUNOLOGY: No

OTHER: reproduction and developmental parameters, plasma thyroid hormone analysis
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 11 days prior to the mating period and during mating until evidence of copulation was observed. Vaginal lavage continued for those
females with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, staging of spermatogenesis
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation. Clinical pathology included measurement of plasma thyroid hormone level.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, according to Guidelines
HISTOPATHOLOGY: Yes, according to Guoidelines
Organ weights according to Guidelines

The numbers of former implantation sites were recorded for all paired females.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Postmortem examinations (offspring):
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/ determined for pups born or found dead.

GROSS NECROPSY
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered
formalin. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or % CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Precoital time = Number of days between initiation of cohabitation and confirmation of mating
Fertility 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
Offspring viability indices:
Post-implantation survival index (%) = (Total number of offspring born/Total number of uterine implantation sites) x 100
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
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
Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Lactation index (%) = (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Salivation seen among animals treated with the test item was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality occurred in two incidences only; the premature death of male no. 26 (300 mg/kg) and female no. 80 (1000 mg/kg) was considered unrelated to treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Compared to controls, body weight gain of male rats was reduced, to about the same extent, at 300 and 1000 mg/kg from start treatment onwards, resulting in about 10% lower mean body weights from treatment Day 36 onwards. Statistical significance was achieved in most weeks of the pre-mating period at 1000 mg/kg and occasionally at 300 mg/kg. To a lesser extent, a similar trend was noted in males treated at 100 mg/kg.
Body weights and body weight gain of females were considered not to be affected by treatment. The slightly higher mean body weights noted in 1000 mg/kg females at most time points (up to 6%, not statistically significant) were considered to represent normal biological variation and, therefore, regarded as unrelated to treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption (before and after correction for body weight) was considered not to be affected by treatment.
For females at 1000 mg/kg a statistically significant decreased relative food consumption was noted on Days 4-7 of lactation. This was caused by the lower food consumption of female no. 75, which was related to her small litter size (i.e. one pup).
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment.
The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significantly lower mean number of platelets was noted at 1000 mg/kg in males (relative difference from controls 17%). Individual values in 1000 mg/kg males generally remained within the historical control range .
There were no treatment-related changes in red and white blood cell parameters. The few statistically significant variations noted in females (higher mean values for total white blood cells, lymphocytes and red blood cell distribution width at 100 or 300 mg/kg) were considered unrelated to treatment due to the lack of a dose-related response.

A statistically significantly higher mean prothrombin time (PT) was noted at 1000 mg/kg in males (relative difference from controls 5%). Values in 1000 mg/kg males remained within the historical control range (and mostly also in the concurrent control range). There were no treatment-related changes in activated partial thromboplastin time (APTT).

Historical control data for the number of platelets (10E9/L) in male Wistar Han rats
Mean: 741, P5- 95: 566–900 (n=91; period 2014 - July 2016)
Historical control data for prothrombin time (s) in male Wistar Han rats
Mean: 17.1, P5-P95: 15.6-18.9 (n=91, period 2014 - July 2016)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes, all at 1000 mg/kg in males, distinguished treated animals from control animals. Relative changes in mean values compared to the control group are indicated between parentheses.
• Higher (26%) plasma activity of aspartate aminotransferase (ASAT)
• Higher (55%) plasma activity of alkaline phosphatase (ALP).
• Higher albumin (7%)
• Higher total bilirubin (50%)
• Lower cholesterol (31%).
Mean values for ASAT, ALP and bilirubin in treated males remained within the historical control ranges, those for albumin and cholesterol were slightly out of these ranges .
The other (mostly statistically significant) variations noted in clinical biochemistry values were considered unrelated to treatment due to the lack of a dose-related response (albumin, creatinine and sodium in 100 and/or 300 mg/kg treated males) or an abnormal value in a single animal (high bile acids in male no. 40 at 1000 mg/kg; values for other liver-related clinical chemistry parameters were also relatively high in this male). Lower ASAT levels in treated females (all dose groups) was related to the relatively high mean control value, due to an abnormal high values in two control females (no. 45 and 49).

Historical control data for male Wistar Han rats (n=94, period 2014 – July 2016)
ASAT (U/L) mean: 75.2, P5-P95: 63.3-92.5
ALP (U/L) mean: 137, P5-P95: 82-246
Albumin (g/L) mean: 32.1, P5-P95: 30.8-33.9
Total bilirubin (umol/L) mean: 2.2, P5-P95: 1.6-3.0
Cholesterol (mmol/L) mean: 1.98, P5-P95: 1.57-2.41

Serum T4 levels in F0-males and F0-females were statistically significantly reduced (by 57% and 19%, respectively) at 1000 mg/kg. The statistically significantly lower (about 20%) mean T4 value noted in males at 100 mg/kg was considered unrelated to treatment due to the lack of a dose response (mean T4 at 300 mg/kg was similar to the control mean). Moreover, individual T4 values at 100 mg/kg generally remained within the concurrent and historical control range .
There were no treatment-related changes in serum TSH levels of both F0-males and F0-females. Overall, mean values of males and females at 100 and 300 mg/kg and males at 1000 mg/kg appeared higher than concurrent control mean values. Due to the relatively high standard deviations of these groups and a lack of a dose response, the higher mean values of the treated animals were considered as not toxicologically relevant. The statistically significantly higher (about four times higher than the control value) mean TSH value noted at 300 mg/kg was considered unrelated to treatment as the increase was caused by abnormal values in two single males (nos. 22 and 29).

Historical control data for T4 values in Wistar Han rats from OECD 422 studies
Males mean: 4.65, P5-P95: 2.970-6.440 (n=448; period 2015-2017)
Females mean: 3.57, P5-P95: 2.183-5.250 (n=40; period 2015-2017)
Note: males within historical control data were 14 weeks, whereas males within the current study were about 19 weeks old at time of blood sampling.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength was considered not to be affected by treatment. The statistically significant differences noted in hind limb grip strength (lower mean values in males at 100 and 1000 mg/kg and in females at 300 mg/kg) showed no clear dose-related response and were not accompanied by corroborative changes in other measures in the neuromuscular domain (including fore limb grip strength, gait, static righting reflex and motor activity). Moreover, the difference from controls was slight and all values remained within normal limits. Therefore, these findings were regarded as unrelated to treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the thyroid gland, an increased incidence of follicular cell hypertrophy was present in 1000 mg/kg females (up to slight).
In the urinary bladder, an increased incidence of hyperplasia/hypertrophy of the urothelium was present in 1000 mg/kg females (minimal). The urothelial change was characterized by a mixture of hyperplasia of the basal cell layer and/or hypertrophy of the dome-shaped surface cells (umbrella cells). The cells of the urothelium formed a uniform thickness (diffuse) without prominent focal outward or inward growth and without cellular atypia. There was no inflammation or cell death involved in this process.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A finding of note was observed in an ovary of 1000 mg/kg/day treated female (no.78) consisting of a tubular adenoma (also known as tubulostromal adenoma). Since there were no other test item-related proliferative findings observed in the ovaries, this incidental finding was considered as unrelated to the test item.
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment.
Most females had regular cycles of four days. Irregular cycles occurred in two females at 100 mg/kg (no. 56 which was not pregnant and no. 57 with a normal litter), and one female at 300 mg/kg was acyclic with extended di-estrus (no. 67 which was not mated; abnormal cyclicity was also indicated by the morphology of her ovaries and vagina). These findings were not attributed to treatment due to their incidental occurrence and lack of a dose-related trend.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
One female at 300 mg/kg showed no evidence of mating (no. 67, mated with male no. 27) and two females were not pregnant despite evidence of mating (no. 46 of the control group and no. 56 of the 100 mg/kg group, mated with male nos. 06 and 16, respectively.
Female no. 67 showed slight vacuolation and hypertrophy of the corpora lutea of the ovaries and a marked increase in mucification of the vagina epithelium indicative of an abnormal cycle. This was consistent with the abnormal estrous cyclicity shown by the vaginal lavage data (female no. 67 was acyclic with persistent di-estrus). This was considered as an incidental finding and unrelated to treatment with the test item. For the two other couples, no abnormalities were seen in the reproductive organs which could account for their non-pregnancy.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
In females treated at 1000 mg/kg microscopic examination revealed an increased incidence of follicular cell hypertrophy (minimal or slight) in the thyroid. This was accompanied by a decrease of thyroid hormone T4 (on average 19%). In males treated at 1000 mg/kg serum levels of thyroid hormone T4 were also decreased (on average by 57%), unaccompanied by treatment related changes in thyroid weight or morphology. For both males and females no corroborative findings were observed in TSH levels. Based on the minimal or slight increase in follicular cell hypertrophy in combination with a decrease in T4 levels in 1000 mg/kg treated females and the magnitude of decreased T4 levels in 1000 mg/kg treated males, adversity could not be excluded.
Exposure to the test item up to 1000 mg/kg was not associated with obvious toxicity in the remaining parental parameters examined in this study (i.e. mortality, clinical appearance, functional tests, ophthalmoscopy, body weight, food consumption, clinical pathology, macroscopic examination and organ weights). Other changes noted in treated rats, mostly at 1000 mg/kg, were considered non-adverse as discussed below.
Slight salivation observed after dosing among treated animals (all dose levels) was considered a physiological response rather than a sign of systemic toxicity.
Male rats administered the test item showed reduced body weight gain from start treatment onwards, which was not accompanied by reduced food consumption. This occurred to a similar degree at 300 and 1000 mg/kg, and to a lesser degree at 100 mg/kg. The resulting reduction in mean body weights was modest (about 10% from Day 36 onwards at 300 and 1000 mg/kg) and the growth rate of treated males had returned to control levels after five weeks of treatment. Therefore, the decreased body weights of treated male rats were regarded as non-adverse within the context of this study.
A further treatment-related morphological change observed in females treated at 1000 mg/kg consisted of an increased incidence of minimal hyperplasia/hypertrophy of the urothelium of the urinary bladder. There was no inflammation or cell death involved in this process. Therefore, this minimal change was considered as non-adverse (Frazier et al., 2012).
Clinical pathology showed several changes in males treated at 1000 mg/kg: higher values for aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total bilirubin and albumin; and lower values for cholesterol. Additionally, the increased prothrombin time (PT) for males at 1000 mg/kg could be related to the decrease in the number of platelets. These changes were not accompanied by adverse anatomic pathology alterations and the mean values for these parameters in 1000 mg/kg males generally remained within the range considered normal for rats of this strain and age (see results section). As such, these clinical pathology changes were regarded as non-adverse (Palazzi et al., 2016).

Frazier, KS, Seely, JC, Hard, GC, Betton, G, Burnett, R, Nakatsuji, S, Nishikawa, A, Durchfeld-Meyer, B, Bube A (2012). Proliferative and Non-proliferative Lesions of the Rat and Mouse Urinary System. Toxicological Pathology 40 (4) suppl., 54S-55S.

Palazzi X, Burkhardt JE, Caplain H, Dellarco V, Fant P, Foster JR, Francke S, Germann P, Gröters S, Harada T, Harleman J, Inui K, Kaufmann W, Lenz B, Nagai H, Pohlmeyer-Esch G, Schulte A, Skydsgaard M, Tomlinson L, Wood CE, Yoshida M (2016). Characterizing "Adversity" of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop. Toxicological Pathology 44(6), 810-24.
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) at 1000 mg/kg appeared lower than that at the other dose levels (9.8 versus 11.4, 12.2 and 11.2 at 0 (control), 100 and 300 mg/kg, respectively). The difference was not statistically significant and could largely be explained by the low number of live pups in a single litter (at first litter check, 1000 mg/kg female no. 75 had 2 live pups and 7 dead pups). Such a small live litter size occasionally occurs in untreated controls. All other litters at 1000 mg/kg had normal live litter sizes . Therefore, the apparently lower mean live litter size at 1000 mg/kg was considered not to reflect an adverse effect of the test item.

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) at 1000 mg/kg was relatively low compared to the concurrent control level (84% versus 89%). Also compared to historical control data, the calculated post-implantation survival index at 1000 mg/kg was at the lower limit of the normal range .
In the nine pregnant females treated at 1000 mg/kg, the individual difference between number of implantation sites and number of pups born (the so-called unaccounted for (implantation) sites) varied between 0 and 4, were 4/9 females had 3, 1/9 had 4 and 4/9 had 0-2 unaccounted for sites, respectively. For comparison, in the nine pregnant control group females 1/9 females had 3 and 8/9 females had 0-2 unaccounted for sites. These results indicated a trend of increased number of females with a higher number of unaccounted for sites, outside the range of that observed in concurrent controls, at 1000 mg/kg.
For female no. 70 (300 mg/kg) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) at 1000 mg/kg (91%) was lower than that at the other dose levels (100, 100 and 99% at 0 (control), 100 and 300 mg/kg, respectively). At first litter check, one pup at 300 mg/kg (no. 66) and 9 pups at 1000 mg/kg (one of litter nos. 71 and 79, seven of litter no. 75) were found dead.

Viability indices (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment up to 300 mg/kg. The viability indices were 100, 98, 100 and 95% at 0 (control), 100, 300 and 1000 mg/kg, respectively.
One pup at 100 mg/kg (litter no. 52) and four pups at 1000 mg/kg (litter nos. 75, 78 and 79) went missing (presumably cannibalized) between PND 2-4. One pup at 300 mg/kg (litter no. 66) and nine pups at 1000 mg/kg (litter nos. 71, 75 and 79) were found dead on PND 1. In addition, one pup at 300 mg/kg was sacrificed early on PND 4, based on its poor physical condition (i.e. lean appearance). At 1000 mg/kg, the postnatal loss differed statistically significantly from that in the control group, in which no pups died. Also compared to historical control data the postnatal loss at 1000 mg/kg was higher .

Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, mean pup weights on PND 1 were 9% (male pups) or 8% (female pups) lower compared to controls. Combined mean pup weights at 1000 mg/kg were statistically significantly lower, however remained within the normal range . Mean pup weights at 1000 mg/kg remained slightly lower at PND 4 and 7, but were close to control values at PND 13. A finding of note was the abnormally low birth weight and severely reduced postnatal growth of the single surviving female pup of 1000 mg/kg female no. 75. Based on the incidental occurrence, the reduced growth of this pup was regarded not toxicologically significant.
Pup weights at 100 and 300 mg/kg showed no remarkable differences from control weights.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were considered unaffected by treatment.
Urinalysis findings:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the findings noted (i.e. alopecia) remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Description (incidence and severity):
The sex ratio of living pups at first litter check in the 1000 mg/kg group differed statistically significantly from that in the control group (% males/females: 34/66 at 1000 mg/kg versus 57/43 in the control group). On an individual litter basis, at 1000 mg/kg the percentage of female pups was about 70-80% in 5/8 litters of normal sizes and 100% in the litter that included only two live pups. For comparison, in the control group the percentage of female pups was about 80% in 1/9 litters and 55% or lower in the remaining litters. The sex ratios at 100 and 300 mg/kg were unremarkable (55/45 and 45/55, respectively).

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
A statistically significantly higher mean value for normalized anogenital distance in female pups at 1000 mg/kg was observed when compared to mean control value (0.59 vs. 0.53, respectively). As the mean values remained within historical control data , the difference between mean control and mean 1000 mg/kg values was minimal and due to the lack of a dose-related response, the increased mean value at 1000 mg/kg was not attributed to treatment.


Treatment up to 1000 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).
No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs).

A skewed sex ratio towards females was noted at 1000 mg/kg: 66% female pups versus 43% in the control group (the difference was statistically significant). With such a high proportion of female pups it could not be excluded that the skewed sex ratio was related to treatment. This was strengthened by the finding that the percentage of female pups was about 70-80% in 5/8 litters of normal size at 1000 mg/kg versus 1/9 in the control group. There were no treatment-related changes in nipple retention in male pups, anogenital distances in male and female pups, or reproductive organs of parental animals. Therefore, it was considered unlikely that the skewed sex ratio resulted from an endocrine mediated (antiandrogenic) effect. An alternative explanation for the skewed sex ratio could be selective loss of male embryos due to a sex difference in sensitivity to toxic effects of the test item. The slightly increased number of unaccounted for implantation sites noted at 1000 mg/kg (resulting in a slightly lower live litter size) might reflect higher sensitivity of male embryos but does not constitute conclusive evidence of selective loss of male embryos.
Live birth index at 1000 mg/kg was lower compared to controls (91% versus 100%). In total, 9 pups of the 1000 mg/kg group were found dead at first litter check: 7/9 of one litter (2 male and 5 female pups) and one male pup in two other litters. Additionally, a slight increase in postnatal loss was noted: 4 pups in three litters died between PND 1 and 4. Furthermore, decreased mean pup weights were noted at 1000 mg/kg on PND 1 (statistically significant for combined mean weights, relative to control 11%). Mean pup weights at 1000 mg/kg remained slightly lower at PND 4 and 7, but were close to control values at PND 13. Based on the combination of a decreased live birth index, a slight increase in postnatal loss and decrease mean pup weights at PND 1, a treatment-related effect on the early viability of the pups could not be excluded.
No treatment-related relevant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation index and duration, lactation indices, parturition, maternal care and the postnatal pup parameters clinical signs, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum concentration of thyroid hormone T4 (PND 13-15) and macroscopy).
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: sex ratio, live birth index, postnatal loss
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: sex ratio, birth index, postnatal loss
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes
Key result
Reproductive effects observed:
no
Conclusions:
Based on decreased T4 levels (for females in combination with increased incidence of follicular cell hyperthrophy in the thyroid) at 1000 mg/kg bw/day, a NOAEL of 300 mg/kg bw/day is derived for parental toxicity.
No fertility effects were observed, therefore, the reproduction NOAEL is concluded to be at least 1000 mg/kg bw/day.
Based on a skewed sex ratio towards females, a reduced live birth index, increased postnatal loss and decreased body weights at 1000 mg/kg bw/day, a NOAEL of 300 mg/kg bw/day is derived for developmental toxicity.
Executive summary:

Wistar Han rats were treated with Ethylene glycol dibenzoate by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, polyethylene glycol 400, alone. Males were treated for 8 weeks prior to mating, during mating, and up to termination (for 92 days). Females that delivered offspring were treated for 8 weeks prior to mating, during mating, duringpost-coitum, and 14-15 days of lactation (for 93-98 days). Females without offspring were treated for 85 days (two non-pregnant females) or 96 days (one female without evidence of mating).

Formulation analysis showed that the formulations were prepared accurately and homogeneously.

Parental results

In females treated at 1000 mg/kgmicroscopic examination revealed an increased incidence of follicular cell hypertrophy (minimal or slight) in the thyroid. This was accompanied by a decrease of thyroid hormone T4 (on average 19%). In males treated at 1000 mg/kg serum levels of thyroid hormone T4 were also decreased (on average by 57%), unaccompanied by treatment related changes in thyroid weight or morphology. For both males and females no corroborative findings were observed in TSH levels. Based on the minimal or slight increase in follicular cell hypertrophy in combination with a decrease in T4 levels in 1000 mg/kg treated females and the magnitude of decreased T4 levels in 1000 mg/kg treated males, adversity could not be excluded.

Exposure to the test item up to 1000 mg/kg was not associated with obvious toxicity in the remaining parental parameters examined in this study (i.e. mortality, clinical appearance, functional tests, ophthalmoscopy, body weight, food consumption, clinical pathology, macroscopic examination and organ weights). Other changes noted in treated rats, mostly at 1000 mg/kg, were considered non-adverse as discussed below.

Slight salivation observed after dosing among treated animals (all dose levels) was considered a physiological response rather than a sign of systemic toxicity.

Male rats administered the test item showed reduced body weight gain from start treatment onwards, which was not accompanied by reduced food consumption. This occurred to a similar degree at 300 and 1000 mg/kg, and to a lesser degree at 100 mg/kg. The resulting reduction in mean body weights was modest (about 10% from Day 36 onwards at 300 and 1000 mg/kg) and the growth rate of treated males had returned to control levels after five weeks of treatment. Therefore, the decreased body weights of treated male rats were regarded as non-adverse within the context of this study.

A further treatment-related morphological change observed in females treated at 1000 mg/kg consisted of anincreased incidence of minimal hyperplasia/hypertrophy of the urothelium of the urinary bladder. There was no inflammation or cell death involved in this process. Therefore, this minimal change was considered as non-adverse (Frazieret al., 20121).

Clinical pathology showed several changes in males treated at 1000 mg/kg: higher values for aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total bilirubin and albumin; and lower values for cholesterol. Additionally, the increased prothrombin time (PT) for males at 1000 mg/kg could be related to the decrease in the number of platelets. These changes were not accompanied byadverse anatomic pathology alterations and the mean values for these parameters in 1000 mg/kg males generally remainedwithin the range considered normal for rats of this strain and age (see results section). As such, these clinical pathology changes were regarded as non-adverse (Palazziet al., 20162).

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs).

Developmental results

A skewed sex ratio towards females was noted at 1000 mg/kg: 66% female pups versus 43% in the control group (the difference was statistically significant due to reverse skewed sex-ratio in the control. Evaluations should have been for each group against historical control cq 50/50 expectation!). With such a high proportion of female pups it could not be excluded that the skewed sex ratio was related to treatment. This was strengthened by the finding that the percentage of female pups was about 70-80% in 5/8 litters of normal size at 1000 mg/kg versus 1/9 in the control group. There were no treatment-related changes in nipple retention in male pups, anogenital distances in male and female pups, or reproductive organs of parental animals. Therefore, it was considered unlikely that the skewed sex ratio resulted from an endocrine mediated (antiandrogenic) effect. An alternative explanation for the skewed sex ratio could be selective loss of male embryos due to a sex difference in sensitivity to toxic effects of the test item. The slightly increased number of unaccounted for implantation sites noted at 1000 mg/kg (resulting in a slightly lower live litter size) might reflect higher sensitivity of male embryos but does not constitute conclusive evidence of selective loss of male embryos. Comparison between ‘Total number of offspring born’ and ‘Total number of uterine implantation sites’ shows a rather comparable loss after implantation of 11.2% in control and 15.7% at 1000 mg/kg bw.

Live birth index at 1000 mg/kg was lower compared to controls (91% versus 100%). In total, 9 pups of the 1000 mg/kg group were found dead at first litter check: 7/9 of one litter (2 male and 5 female pups) and one male pup in two other litters. Additionally, a slight increase in postnatal loss was noted: 4 pups in three litters died between PND 1 and 4. Furthermore, decreased mean pup weights were noted at 1000 mg/kg on PND 1 (statistically significant for combined mean weights, relative to control 11% - however, as male and female pups differ in BW, the skewed sex-ratio with high proportion of females causes contributes to the difference. BW can only be compared for male and female pups separately.). Mean pup weights at 1000 mg/kg remained slightly lower at PND 4 and 7, but were close to control values at PND 13. The report concludes that based on the combination of a decreased live birth index, a slight increase in postnatal loss and decrease mean pup weights at PND 1, a treatment-related effect on the early viability of the pups could not be excluded. Attached are graphs of the development of the BW relative to the control during PND 1 to 13 for male respectively female pups. The 1000 mg/kg group shows a lower BW on PND 1 compared to the control, but as can be seen, it as rather similar for the 100 mg/kg group, whereas the 300 mg/kg is the same as control. The clear lack of a dose-response indicates that the observed difference is likely for a large part just fortuitous.

 

No treatment-related relevant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation index and duration, lactation indices, parturition, maternal care and the postnatal pup parameters clinical signs, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum concentration of thyroid hormone T4 (PND 13-15) and macroscopy).

Endpoint:
developmental neurotoxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2020 - May 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 426 (Developmental Neurotoxicity Study)
Version / remarks:
October 2007
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Stability in polyethylene glycol 400: Stability for at least 5 hours at room temperature under normal laboratory conditions and 8 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Females: time-mated, arrived on day 0 (day of successful mating) or day 1 post-coitum
- Age at study initiation: 10-15 wks
- Weight at initiation of dosing: 188-257 g
- Fasting period before study: no
- Housing: On arrival, females were individually housed in Makrolon plastic cages (MIII type, height 18 cm) until necropsy.
Pups were housed with the dam until weaning (PND 21; Subsets B-D), necropsy (Subset A, PND 21-22), or until necropsy of the dam (surplus pups). Subsets are described below at 'Details on study design'.
Subset B-D animals were housed in groups of maximum 5 animals/sex/cage in Makrolon cages (MIV type, height 18 cm).
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water for a maximum of 2 hours.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water: Municipal tap water was freely available to each animal via water bottles.
During motor activity measurements, animals had no access to food and water for a maximum of 2 hours. Analyses of food and water were available. It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 43-79
- Air changes (per hr): at least 10 (100% fresh air, no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 22 Aug 2020 To: 16 Nov 2020
Route of administration:
oral: gavage
Vehicle:
other: polyethylene glycol 400
Details on exposure:
RATIONALE FOR ROUTE OF EXPOSURE
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

PREPARATION OF DOSING SOLUTIONS
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a suspension and were heated to a maximum temperature of 60±5°C for at least 30 minutes to obtain visual homogeneity. Formulations were filled out in daily portions. If dosed on the day of formulation, formulations were released for dosing when they obtained a temperature of 40°C or lower. If formulations were not dosed on the day of formulation, they were stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 5 hours after removal from the refrigerator.

ADMINISTRATION
The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The dosing formulations were stirred continuously during dose administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial preparations
- Concentration in vehicle: 20, 60 or 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
Time-mated females were received at the test facility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Trial formulations (not used for dosing) were prepared at target concentration levels of 20 mg/mL (low) and 200 mg/mL (high) and analyzed according to a validated analytical procedure. Analysis of accuracy of preparation and homogeneity was performed at the top, middle and bottom of the containers.
The concentration (all groups) and homogeneity of the dose formulations (low and high dose) were analysed in week 1 of treatment (duplicate sets of samples).
The accuracy of preparation was considered acceptable if the mean analyzed concentrations were 90-110% of the target concentrations. Homogeneity is demonstrated if the coefficient of variation is ≤ 10%.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 516416) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
from post-coitum day 6 up to and including lactation day 20
Frequency of treatment:
once daily, 7 days per week
Details on study schedule:
- Time-mated females were used.
- F1-animals were not dosed directly, since in a Lactational Transfer Study (Test Facility Study No. 20218705) it was demonstrated that test item was excreted into maternal milk from lactating dams exposed to 1000 mg/kg bw/day from Day 6 post-coitum to Day 13 of lactation, inclusive.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
F0: 25 females
F1: 80 animals/dose/sex divided into 4 subsets (A, B, C and D)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on previous studies conducted with Ethylene glycol dibenzoate in rats including a Combined 90-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422, Test Facility Study No. 516410), a Lactational Transfer Study (Test Facility Study No. 20218705) and a Prenatal Developmental Toxicity Study (OECD 414, Test Facility Study No. 20218707).
In the combined 90-Day study (Test Facility Study No. 516410), Wistar Han rats received 0, 100, 300 or 1000 mg/kg bw/day by daily oral gavage. A parental NOAEL of 300 mg/kg bw/day was established based on decreased total T4 levels (on average 57% and 19% for males and females, respectively; for females in combination with increased incidence of (minimal or slight) follicular cell hypertrophy in the thyroid) at 1000 mg/kg bw/day. A developmental NOAEL of 300 mg/kg bw/day was established based on a skewed sex ratio towards females (66% female pups versus 43% in the control group), a reduced live birth index (91% vs. 100% in the control group), increased postnatal loss (viability index of 95% vs. 100% in the control group) and decreased body weights on PND 1 at 1000 mg/kg bw/day (11% lower than the control group). No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day). In this study, males and females were treated for 8 weeks prior to mating, whereas females in a developmental neurotoxicity study will be treated from Day 6 post-coitum up to and including Day 20 of lactation.
In the Lactational Transfer Study (Test Facility Study No. 20218705), no maternal toxicity was recorded for Wistar Han rats receiving 1000 mg/kg bw/day by daily oral gavage from Day 6 post-coitum to Day 13 of lactation.
In the Reproduction/Developmental Toxicity Screening Test Wistar Han rats received 0, 100, 300 or 1000 mg/kg bw/day by daily oral gavage. A Maternal NOAEL of at least 1000 mg/kg bw/day was established, and a Developmental NOAEL of 300 mg/kg/day was established based on lower fetal body weights observed at 1000 mg/kg bw/day (mean combined fetal body weights showed a relative difference to concurrent controls of -8%).
Overall, these data indicated that a high dose level of 1000 mg/kg bw/day was expected to be tolerated in a developmental neurotoxicity study. The high-dose level should produce some maternal and/or developmental toxic effects, but not death nor obvious suffering. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- F1 animal assignment: On PND 4, eight pups from each litter of equal sex distribution (the first four males and first four females per litter, if possible) were selected to reduce variability among the litters. The non-selected pups were culled on PND 4.
Allocation of F1-pups for behavioural tests and neurohistopathological examination:
On PND 1, or shortly thereafter, one pup/sex/litter (if possible) was assigned to Subsets A, B, C and D, to have a total of 20 pups selected per sex per group per subset. A total of 20 females with 8 live pups/litter were selected (if possible).

Subset A:
• Neurohistopathological and morphometric evaluation of brain (Groups 1 and 4) and brain weights (PND 21-22)

Subset B:
• Behavioral ontogeny (righting reflex; beginning on PND 2)
• Locomotor activity (PND 13, 17, 25± 2, and 60±2)
• Neurohistopathological evaluation of central nervous system (CNS) and peripheral nervous system (PNS), morphometric evaluation of brain (Groups 1 and 4) and brain weights (PND 70-73)

Subset C:
• Sexual maturation: vaginal patency (from PND 25 onwards) and balanopreputial separation (from PND 35 onwards)
• Acoustic startle (PND 25±2 and 60±2)
• Biel Maze (PND 23-27)

Subset D:
• Detailed clinical (arena) observations (PND 25±2, 35± 2, 45± 2 and 60± 2)
• Biel Maze (PND 65-70)
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at least once daily, from day 6 post-coitum onwards up to the day prior to necropsy

BODY WEIGHT:
- Time schedule for examinations: Days 6, 9, 12, 15, 18 and 21 post-coitum, and on PND 1, 4, 7, 11, 14, 17 and 21 (weaning)

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, over Days 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum, and over PND 1-4, 4-7, 7-11, 11-14, 14-17 and 17-21

WATER CONSUMPTION: monitored on regular basis throughout the study by visual inspection of the water bottles

OTHER:
Detailed Arena Observations were conducted on post-coitum Days 8 and 17 and lactation Days 7 and 14 for 10 dams/group (the first 2 or 3 sequential animal Nos. per mating date). These observations were conducted prior to dosing, approximately the same time each day and with a maximum of 3 hours difference between the earliest and latest observation.
For the detailed functional observations, testing of animals was counterbalanced across dose groups to the extent possible, based on the number of animals available for each delivery date. These observations were conducted by technicians not involved in dosing or regular observations of these animals.
Animals were transferred to the separate room equipped for these purposes on the day preceding the day on which detailed functional observations were conducted:
1. Detailed clinical observations consisted of a number of tests conducted in- and outside the home cage. To the extent possible, testing of animals was counterbalanced across dose groups. The clinical observations took place in a quiet room.
2. Rectal temperature was measured immediately after the detailed clinical observations.
3. Hearing ability (Score 0 = normal/present, score 1 = abnormal/absent).
4. Pupillary reflex (both eyes; Score 0 = normal/present, score 1 = abnormal/absent).

GENERAL REPRODUCTION DATA
For each female, confirmation of pregnancy and delivery day were recorded.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Litter observations:
STANDARDISATION OF LITTERS: see under details of study design

The following parameters were examined in the F1 generation:
MORTALITY/MORIBUNDITY:
Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Pups showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

CLINICAL OBSERVATIONS:
Clinical observations were performed at least once daily (home cage). For all Subset D animals, a hand-held observation was additionally conducted on PND 7 and 14.

BODY WEIGHT:
Live pups were weighed individually on PND 1, 4, 7, 11, 14, 17 and 21 (all animals).
From PND 21 onwards, all animals were weighed weekly, from weaning onwards.
Animals of each subset were also weighed on the day of acquisition of righting reflex, balanopreputial separation and vaginal opening, i.e. for Subset B animals on the day of acquiring righting reflex, and for Subset C animals for the first animal per litter for which vaginal opening and balanopreputial separation was reached.

FOOD CONSUMPTION:
Food consumption was quantitatively measured weekly, starting from within one week after all animals were weaned.

WATER CONSUMPTION:
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles. No quantitative investigation was introduced as no effect was suspected.

FUNCTIONAL AND BEHAVIORAL TESTS:
Detailed Arena Observations:
Detailed Arena Observations were conducted on PND 25±2, 35±2, 45±2 and 60±2, for all Subset D animals. Observations/measurements were conducted in separate room(s) specially equipped for these purposes or in the study room (detailed recorded in the study raw data). When conducted in a separate room, animals were transferred to this room on the day preceding the day on which detailed functional observations were conducted. These observations were conducted approximately the same time each day and with a maximum of 3 hours difference between the earliest and latest observation.
1. Detailed clinical observations consisted of a number of tests conducted in- and outside the home cage. To the extent possible, testing of animals was counterbalanced across dose groups. The clinical observations took place in a quiet room.
2. Rectal temperature was measured immediately after the detailed clinical observations.
3. Hearing ability (Score 0 = normal/present, score 1 = abnormal/absent).
4. Pupillary reflex (both eyes; Score 0 = normal/present, score 1 = abnormal/absent).

Righthing reflex:
Acquisition of the righting reflex was observed daily, beginning on PND 2 for all Subset B animals (20/sex/group) and continued for the respective pups until positive.

Acoustic startle response:
Acoustic startle response (habituation) was assessed using the StartleMonitor System (Kinder Scientific, Poway, USA). This was performed on PND 25± 2 and 60± 2, for all Subset C animals (20 animals/sex/group) in a sound-attenuated room.
To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose group and sex. The animals were tested in sets of up to 3. The test sessions consisted of a five-minute acclimation period with a 65 ± 5-dB broadband background white noise. The startle stimulus for each trial was a 115 ± 5-dB mixed frequency noise burst stimulus (approximately 20 milliseconds in duration). Responses were recorded during the first 250 milliseconds following onset of the startle stimulus for each trial. The test session consisted of 50 trials with an eight-second intertrial interval. Average response amplitude (AveN), average maximum response amplitude (MaxN) and average latency to achieving the maximum response amplitude (Tmax) were analyzed in five blocks of 10 trials each.

Locomotor activity:
Locomotor activity was tested on PND 13, 17, 25±2, and 60± 2, for all Subset B animals (20 pups/sex/group) using the Kinder Scientific Motor Monitor System. Recording period was one hour under normal laboratory light conditions.
To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose groups.
Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

Learning and memory test (Biel Maze):
Cognitive function (learning and memory retention) was conducted for all Subset C animals (20 animals/sex/group) between PND 23-27 and for all Subset D pups (20 pups/sex/group) between PND 65-70; “Day 1” as specified below occurred within these respective periods.
A water-filled Biel Maze was used. A total of 4 trials to verify swimming ability and adapt the animals to the water maze conditions, 5 trials for learning and 1 for memory assessment was performed.
The time to escape and number of errors (animal deviates from the correct channel with all four feet) were recorded. Animals were allowed three minutes to escape (except for the straight channel swimming test for which the animals were allowed two minutes) after which they were removed.
Each animal was tested as follows:
Day 1: Four consecutive trials in the straight channel.
Day 2: Five trials in Path A (minimum inter-trial interval of one hour)
Day 9: One trial in Path A.

SEX AND SEXUAL MATURATION:
Sex was externally determined for all pups on PND 1 and 4.
Vaginal patency (vaginal opening) was monitored daily for all females from Subset C (20 rats/group) from PND 25 onwards. until vaginal patency was present, by visual inspection of the vaginal area.
Balanopreputial separation (prepuce opening) was monitored daily for all males from Subset C (20 rats/group) from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area.
Body weight was recorded on the day of acquisition of vaginal patency/balanopreputial separation.

CLINICAL PATHOLOGY
Blood of 10 F1-animals/sex/group (2-3 animals/sex/group per mating date, if possible) from Subset A (non-perfused animals; PND 21-22) and Subset B (PND 70-73) were collected on the day of scheduled necropsy. Animals were not fasted overnight.
Samples were collected using the sequence Group 1-2-3-4 on necropsy Day 1, Group 2-3-4-1 on necropsy Days 2, Group 3-4-1-2 on necropsy Day 3 etc. (if practically and/or logistically feasible). Samples were collected between 7.00 and 10:30 a.m., from the aorta under isoflurane anesthesia as part of the necropsy procedure from Surplus animals at necropsy (PND 24-25) since blood samples collected at necropsy from Subset A animals were not processed into serum and therefore not subjected to thyroid hormone analysis. It should be noted that the F0 animals to which these spare animals belonged were dosed up to and including PND 20.
Blood samples at a target volume of 1.0 mL (Subset A (non-perfused), Surplus (non-perfused) and Subset B animals) were collected into tubes without anticoagulant. Blood samples were processed for serum.
Serum samples retained for possible future analysis of T3, T4 and TSH were maintained by the Test Facility in the freezer (≤-75°C). Under these storage conditions, samples are stable for 6 months.
Postmortem examinations (parental animals):
SACRIFICE AND GROSS NECROPSY
Scheduled necropsy on LD 21-25 for females that delivered, post-coitum day 26 for females which failed to deliver, within 24 hrs after the last pup was found dead or missing for a female with total litter loss. Animals were fasted overnight before necropsy.
All animals were subjected to an external, thoracic and abdominal examination. All macroscopic abnormalities were recorded.
No terminal body weight was recorded and no organs were weighed
Postmortem examinations (offspring):
TERMINAL PROCEDURES
Unscheduled deaths:
Pups that were sacrificed in extremis, younger than 7 days, were euthanized by decapitation.
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology.
Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Culled pups (PND 4):
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation.
Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

Surplus pups:
Animals were not fasted overnight before necropsy.
Surplus pups were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%), and were examined externally and sexed (both externally and internally).
From several Surplus pups (as defined in the study records), a blood sample was collected from the aorta since from a few Subset A animals the collected blood sample was stored at room temperature too long to allow a meaningful analysis of these samples. These pups were deeply anaesthetized using isoflurane prior to blood sampling and were exsanguinated after blood sampling.

Subset A and B:
Animals were not fasted overnight before necropsy.
Subset A and B animals selected for neuropathology were euthanized by whole body (in situ) fixation perfusion.
Subset A and B animals not selected for whole body perfusion were deeply anaesthetized using isoflurane and subsequently exsanguinated.
Necropsy procedures for Subsets A and B animals were counterbalanced across dose groups as much as practically feasible.
Animals selected for neuropathology:
On PND 21-22 (Subset A) or PND 70-73 (Subset B), all animals had a terminal body weight taken. A total of 10 animals/sex/group (first 2-3 animals/sex/group per necropsy date, if possible) per Subset A and B were first anesthetized with isoflurane. The animals were subsequently sacrificed by whole body (in situ) fixation perfusion.
After perfusion, the cranium was removed, exposing the brain. The skull including the brain was placed in 10% buffered formalin and allowed to fix for at least 7 days prior to removal from the skull. The fixed brains were removed and weighed, and the length and maximum width of the brain was measured for all animals selected for neuropathology. Subsequently, the brain was fixed in 10% buffered formalin together with selected PNS tissues.
After sacrifice all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to tissues of the central and peripheral nervous system. Descriptions of all abnormalities were recorded and fixed in 10% buffered formalin. Sex was determined both externally and internally.
Animals selected for unfixed brain weights:
On PND 21-22 (Subset A) or PND 70-73 (Subset B), the remaining animals of Subsets A and B had a terminal body weight taken and were deeply anaesthetized using isoflurane.
A blood sample was subsequently collected from the aorta from all these animals.
Animals were subsequently exsanguinated and subjected to a gross necropsy. The brains were immediately removed, weighed and immersion fixed in 10% buffered formalin.
After sacrifice all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to tissues of the central and peripheral nervous system. Descriptions of all abnormalities were recorded. Abnormalities were fixed in 10% buffered formalin. Sex was determined both externally and internally

Subset C and D:
Animals were not fasted overnight before necropsy.
On PND 63-70 (Subset C) or PND 78-81 (Subset D), all surviving animals were euthanized by an oxygen/carbon dioxide procedure. After sacrifice all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to tissues of the central and peripheral nervous system. Descriptions of all abnormalities were recorded. Abnormalities were fixed in 10% buffered formalin. Sex was determined both externally and internally.

HISTOLOGY
All PNS and CNS tissues (including the skull containing olfactory bulbs) collected from
10 perfusion fixed animals/sex/group of Subset A and B were investigated.
PND 21-22 CNS tissues (Subset A):
For the 10 perfusion fixed animals/sex/group, CNS tissues selected for quantitative histopathology was embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with HE.
For morphometric analysis, 3 consecutive sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections were obtained. These sections of the brain of perfusion-fixed Subset A animals of Groups 1 and 4 initially were also stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet.
PND 70-73 CNS and PNS tissues (Subset B):
For the 10 perfusion fixed animals/sex/group, CNS and PNS selected for quantitative histopathology were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with HE.
At the cervical and lumbar position of the spinal cord a longitudinal (1-1.5 cm) and transversal cut (approx. 3 mm thickness) were made, including the cervical and lumbar swellings. At the position of both the cervical and lumbar swellings, multiple ganglia (if possible) were dissected with the dorsal and ventral nerve roots attached.
The spinal cord and peripheral nerve sections were included both cross or transversal and longitudinal sections.
For morphometric analysis, 3 consecutive sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections were obtained. These sections of the brain of perfusion-fixed Subset B animals of Groups 1 and 4 initially were also stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet.

HISTOPATHOLOGY
- Morphometric (quantitative) analyses of brain tissues was performed for Subset A and B of Groups 1 and 4.
Microscopic morphometric measurements were performed on specified sections of brain. Analyses included measurements from neocortical, hippocampal, and cerebellar areas. Slides were scanned using a Hamamatsu Nanozoomer whole slide scanner and imported into the Visiopharm software for measurements. These measurements were performed in a blinded random fashion for all dose groups.
Statistics:
Data collected/processed in ToxData:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Non-parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
For the startle data set normal distribution was determined by using the Kolmogorov-Smirnov, Cramer-von Mises and Anderson-Darling tests. As the majority of the data was not normally distributed on PND 25 and 60, the Kruskal-Wallis test was applied to determine overall statistical significance.
Incidence: An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Data from Provantis: see "Any other info on mat/method
Reproductive indices:
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
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
Viability index (%): (Number of live offspring on PND 4 before culling/Number live offspring on PND 1 after littering) x 100
Weaning index (%): (Number of live offspring on PND 21/Number of live offspring on PND 4) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were noted that were considered to be related to toxicity of the test item.
Salivation seen after dosing among a few animals at 300 and 1000 mg/kg bw/day on several days during the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
No clinical signs were observed for females at 100 mg/kg bw/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality occurred during the study period.
One control female was sacrificed on post-coitum Day 24 because of total litter loss.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were considered not affected by treatment with the test item.
For one female at 1000 mg/kg bw/day, a notable weight loss was recorded over lactation Days 14-17 along with a reduced food intake. This was ascribed to a dysfunctioning water bottle due to which the animal presumably had no or limited access to water for a maximum of approximately three days. On PND 17, this animal was found to have hunched posture and its pups had piloerection and ptosis as well as lower weight gain between lactation Days 14 and 17. The water bottle was replaced on PND 17 following which this animal gained weight and had normal food intake. Also, no clinical signs were observed for the pups from this day onwards and weight gain resumed to normal values. This incidental occurrence was therefore considered not to have affected the outcome of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Absolute and relative food consumption were considered not affected by treatment with the test item.
For one female at 1000 mg/kg bw/day, a reduced food intake was recorded over lactation Days 14-17. This was unrelated to treatment with the test item as outlined under body weight.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Arena observation parameters as determined on post-coitum Days 8 and 17 and lactation Days 7 and 14 were considered not affected by treatment with the test item. Incidences of observed symptoms did not show a dose-related trend, and/or had incidences/severities that were similar to the control group.
All examined animals had a normal hearing and pupillary reflex, and rectal temperature was similar across the dose groups on all measurement occasions.
Description (incidence and severity):
One female at 100 mg/kg bw/day and one female at 300 mg/kg bw/day did not deliver offspring and were sent for necropsy on post-coitum Day 26. One control female was sent for necropsy on post-coitum Day 24 as this female had total litter loss. This female was recorded to be delivering pups on post-coitum Day 23, but no pups were present during the first litter check on post-coitum Day 24. Therefore, it was not included in the calculation of postnatal loss, live birth index and viability index of the control group. These incidences were considered not to be related to treatment with the test item, as they occurred in the absence of a dose-related trend and may be encountered in this type of study. All other females delivered offspring.
Duration of gestation was considered not to be affected by treatment with the test item.
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Pre-weaning:
No clinical signs occurred among pups that were considered to be related to treatment with the test item .
The nature and incidence of recorded clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item.

Post-weaning:
Post-weaning clinical observations revealed no clinical signs that were considered to be related to treatment with the test item.
Any clinical signs noted after weaning occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend.
At the incidence observed, these were considered to be unrelated to treatment with the test item.
No clinical signs were recorded for control group females.

Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Pre-weaning:
Litter size was considered not affected by treatment with the test item.
Mean litter sizes were 10.6, 10.3, 10.5 and 10.6 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment with the test item. The live birth indices were 100% for the control, 300 and 1000 mg/kg bw/day group each, and 99% for the 100 mg/kg bw/day group.
Two pups at 100 mg/kg bw/day and one pup at 1000 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Viability index (the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1) was considered not to be affected by treatment with the test item.
Viability indices were 100% for the control group, 99% for the 100 and 300 mg/kg bw/day groups, and 98% for the 1000 mg/kg bw/day group.
Two pups each at 100 and 300 mg/kg bw/day and four pups at 1000 mg/kg bw/day were found dead or missing on PND 3 or 4. Two of these missing pups at 1000 mg/kg bw/day felt cold at first litter check. In addition, one pup at 1000 mg/kg bw/day was sacrificed for ethical reasons on PND 1 due to a wound on the right front leg. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item. No pups were found dead/missing between lactation Days 5 and 21, resulting in a weaning index of 100% for all groups.

Post-weaning: no mortality occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, pre-weaning body weights of male and female pups were lower than control means from lactation Day 4 onwards, achieving a level of statistical significance between PND 7 and 17 on most occasions for males and on PND 11 for females (see also clin. signs F0). On lactation Day 21, mean pre-weaning body weight of both sexes combined was 0.96x of control mean.
Body weights of pups at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item.

Post-weaning:
At 300 and 1000 mg/kg bw/day, mean body weight of males was lower than the control means from Day 1 of post-weaning onwards (0.95x of control for both groups), and remained lower during the first four or two weeks of post-weaning respectively (statistically significant). Mean absolute body weight at these dose levels was similar to control means at the end of the study period. This was reflected by a statistically significantly higher body weight gain of males at 1000 mg/kg bw/day from Day 22 of post-weaning onwards (1.07x of control at the end of the study period). Mean body weight gain of males of the 300 mg/kg bw/day group showed no apparent dose-related changes throughout the study period.
Body weight gain of females of the 1000 mg/kg bw/day group was higher than control means during most of the study period (1.06x of control at the end of the study period), but mean absolute body weights remained similar to control means throughout the study period. It should be noted that since body weights on Day 1 for animals of this age are relatively low, rather large changes in weight gain can occur with small increases in absolute body weight.
.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Absolute and relative food consumption were considered not affected by treatment with the test item.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Serum T3, T4 and TSH levels on PND 21-22 and PND 70-73 were considered not to be affected by treatment with the test item.
All variations in these parameters, including the statistically significantly higher total T3 value for males of the 300 mg/kg bw/day group on PND 70-73, occurred in the absence of a dose-related trend.
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio during lactation was not affected by treatment with the test item.
Time to achieving balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening) in females were considered not affected by treatment with the test item.
Mean body weight on the day of achieving balanopreputial separation or vaginal opening
was similar across the dose groups.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Fresh and fixed brain weight were considered not affected by treatment with the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Up to weaning no macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item.

Post-weaning:
There were no test item-related gross observations recorded for Subsets A, B, C and D animals.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings:
no effects observed
Description (incidence and severity):
Brain dimensions (length and width of brain) of Subset A and B animals were considered not affected by treatment with the test item.
There were no microscopic findings in the peripheral and central nervous tissues examined for the control or high dose (1000 mg/kg bw/day) group males or females of Subset A and B after treatment with the test item based on examination of the H&E and Luxol Fast Blue/Cresyl Violet stains.
Morphometric brain measurements for Subset A and B animals were comparable with control values.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Detailed clinical observations:
Arena observation parameters as determined on PND 25, 35, 45 and 60 were considered not affected by treatment with the test item. Incidences of observed symptoms did not show a dose-related trend.
One male of the 100 mg/kg bw/day group had a small right pupil on PND 60 which did not dilate due to an eye lesion, and one female of the 1000 mg/kg bw/day group had a dilated left pupil on PND 60 which did not become smaller . All other animals had a normal pupillary reflex. All animals had a normal hearing reflex and rectal temperature was considered not affected by treatment with the test item. The statistically significantly higher rectal temperature of females of the 1000 mg/kg bw/day group on PND 45 was considered not to be related to treatment with the test item since no such change was apparent on other measurement occasions.

Righting reflex:
The day on which righting reflex was acquired was considered not affected by treatment with the test item. Animals showed - on average - a positive righting reflex on PND 2 or 3.

Acoustic startle response:
Acoustic startle response on PND 25 and PND 60 was considered not affected by treatment with the test item. Means of average and maximum response amplitude and latency to maximum response amplitude remained similar to control means on all occasions.

Motor activity:
Motor activity was considered not affected by treatment with the test item.
PND 13
The lower mean motor activity of males and females in the 1000 mg/kg bw/day group, (0.75x and 0.66x of control for total movements and ambulations, respectively for males and 0.82x and 0.68x of control for total movements and ambulations, respectively for females; not statistically significant) was attributed to high values in three control group animals. When discounting these high control values in the mean calculations, the mean control values were 3238 and 870 counts for total movements and ambulations of males, and 3439 and 1043 counts for total movements and ambulations of females. These mean values were similar to or even lower than the means including these higher individual values. It was therefore considered that these variations in motor activity did not represent a change related to treatment with the test item.
In relation to the above described variations in individual control values, variations in mean motor activity levels across the dose groups did not show a dose-related trend and were therefore considered not to represent an effect of treatment with the test item.
PND 17
Motor activity was considered not affected by treatment with the test item.
PND 25
The higher mean motor activity of males in the 1000 mg/kg bw/day group (1.29x and 1.30x of control for total movements and ambulations, respectively) was ascribed to a lower motor activity of control males during intervals 6, 7 and 8. Also, a clear dose-related trend was absent; motor activity/habituation pattern was similar across all test item-treated groups. Variations of these parameters were therefore considered not to be related to treatment with the test item.
Motor activity of females was considered not affected by treatment with the test item.
All male and female groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
PND 60
Mean motor activity (total movements and ambulations) of females at 100, 300 and 1000 mg/kg bw/day appeared higher than the control means (not statistically significant). In absence of a dose-related trend, these variations were considered not to be related to treatment with the test item.
All male and female groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Learning and memory test (Biel maze):
No effect on learning and memory was observed that was considered to be related to treatment with the test item on PND 23-27 and PND 65-70.
The straight channel trial confirmed swimming ability for all rats. The statistically significantly higher mean time to reach the platform of females of the 100 and 300 mg/kg bw/day groups during the first trial of the straight channel swimming test occurred in the absence of a dose-related trend and was therefore considered not to be related to treatment with the test item. During subsequent swimming trials, the mean time to reach the platform was similar across all groups.
Mean time required to reach the platform and number of errors made during the five consecutive swimming trials in the Biel maze was similar between treated and control animals on PND 23-27 and PND 65-70 and showed an expected downward trend over these trials. Any statistically significant changes occurring during the swimming trials occurred in the absence of a dose-related trend and were therefore considered not to be related to treatment with the test item.
The memory swimming trial conducted seven days after the swimming trials also showed that the mean time required to reach the platform and number of errors made was similar between treated and control animals on PND 23-27. In the memory swimming trial conducted on PND 65-70, the mean time to reach the platform by 1000 mg/kg bw/day group male rats appeared higher than controls (not statistically significant). However, the intra-group variation was high, and on PND 23-27 mean time to reach the platform of these animals was similar to control levels. As such, this variation was considered not to be related to treatment with the test item.
On PND 23-27 and PND 65-70, the memory swimming trial showed no apparent dose-related differences compared to mean values in the last swimming trial conducted seven days earlier.
Key result
Dose descriptor:
NOAEL
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:
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Dose formulation analysis:

Accuracy
In Group 1 (vehicle), no test item was detected.
The concentrations analyzed in the formulations of Groups 2 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
For the formulation of Group 3, the mean accuracy was below the target concentration
(i.e. 89% of target). This result was accepted as it was only 1% below the target range.
Homogeneity
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Conclusions:
In a developmental neurotoxicity study performed in rats according to OECD 426 and GLP principles, the maternal and developmental (including neurotoxicity endpoints) NOAEL for the test item were established to be at least 1000 mg/kg bw/day.
Executive summary:

A developmental neurotoxicity study was performed in rats according to OECD 426 and GLP principles with the test item. Time-mated female rats (25/dose) were given 0, 100, 300 and 1000 mg/kg bw/day from day 6 post-coitum up to and including lactation day 20. 

For the F0-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption and gross necropsy findings.
For the F1-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, sexual maturation (vaginal patency and balanopreputial separation), functional and behavioral tests including detailed clinical observations, righting reflex,, acoustic startle response, learning and memory test (Biel Maze) and motor activity, clinical biochemistry (T3, T4, TSH), gross necropsy findings, brain weights and dimensions and histopathologic examination of central and peripheral nervous tissues (including brain morphometry).
In addition, the following reproduction/developmental parameters were determined for the F0-generation: gestation duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex and macroscopy).
Formulation analyses confirmed that formulations of test item in polyethylene glycol 400 were prepared accurately and homogenously.

F0-generation
Enlargement of the liver was recorded for two animals at 1000 mg/kg bw/day and for one animal at 300 mg/kg bw/day. This necropsy finding could not be supported by other morphological changes since liver weight and histopathology were not part of this study. Although a relationship to treatment could not be excluded, its low incidence was considered to support the non-adversity of this finding.
No treatment-related changes were noted in clinical appearance, body weight and food consumption, and no test item-related mortality occurred during the study period.

F1-generation – pre-weaning
A non-adverse lower mean pre-weaning body weight was recorded at 1000 mg/kg bw/day, for both sexes from lactation Day 4 onwards (0.96x of control mean on lactation Day 21, for both sexes combined). Body weights of pups at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item. Since this body weight variation was slight and other pre-weaning developmental parameters remained unaffected by treatment with the test item, this was considered not adverse.

No test item-related changes were noted in any of the other pre-weaning developmental parameters investigated in this study (i.e. duration of gestation, parturition, sex ratio, maternal care, litter size, live birth index, viability index, weaning index, clinical signs and macroscopy).

F1-generation - post-weaning
A non-adverse lower mean body weight and/or higher weight gain was recorded for males and/or females during the initial weeks of post-weaning in the 300 and 1000 mg/kg bw/day groups, but absolute body weights attained values similar to control levels during the study period.
No test item-related changes were noted in any of the other F1-generation
post-weaning developmental parameters investigated in this study (i.e mortality, clinical signs, food consumption, balanopreputial separation, vaginal opening, total T3, T4 and TSH levels (See attached graphs) and macroscopy).

F1-generation - neurotoxicity results
No test item-related changes were noted in any of the F1-generation/
(neuro-)developmental parameters investigated in this study (i.e. time to acquire righting reflex, arena observations, hearing and pupillary reflex, rectal temperature, learning and memory, acoustic startle response, motor activity, gross brain dimensions, brain weights, brain morphometry, and peripheral and central nervous tissue histopathology).
In conclusion, based on the results of this Developmental Neurotoxicity Study, the following No Observed Adverse Effect Levels (NOAEL) of Ethylene Glycol Dibenzoate were established:

Maternal NOAEL: at least 1000 mg/kg bw/day

Developmental (including neurotoxicity endpoints) NOAEL: at least 1000 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP and OECD guideline K1 studies are 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

Combined repeated dose (90-day) and reproduction toxicity screening (OECD 422):

10 rats/sex/group received 0, 100, 300 and 1000 mg/kg Ethylene glycol dibenzoate (EGDB) by daily oral gavage for 8 weeks prior to mating, during mating, and up to termination (for 92 days males) after 14-15 days of lactation (for 93-98 days females).

Parental results:

Based on decreased T4 levels (for females in combination with increased incidence of follicular cell hypertrophy in the thyroid) at 1000 mg/kg, of which adversity could not be excluded, a NOAEL of 300 mg/kg bw/day was derived for parental toxicity. All other parameters were without significant findings.

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs).

 

In order assess the possible hazard for human thyroid insufficiency in adults as well as pre- and post-natal neurological development of offspring, a developmental neurotoxicity study (OECD 426) has been performed.Female rats were dosed at 0, 100, 300 and 1000 mg EGDB/kg bw in 25 female rats per dose group from gd 6 through to lactation day 20. Pups were not dosed directly; excretion into maternal milk from lactating dams was demonstrated in a lactational transfer study.

On PND 4 pups were culled to 8 per litter, and assigned 1 pup/sex/litter to 4 sub-sets (A,B,C and D) to make up 20/sex/grp for each of the four sub-sets.

Included were observations to detect gross neurologic and behavioural abnormalities, including the assessment of physical development, behavioural ontogeny, motor activity, motor and sensory function, and learning and memory and the evaluation of brain weights and neuropathology during postnatal development and adulthood.

Results:

F0-generation: Besides liver enlargement observed in 2 HD and 1 MD female, which was considered to be non-adverse, no maternal toxicity was observed. There were no effects observed on reproduction parameters.

F1-generation, pre-weaning: A non-adverse slightly lower mean pre-weaning body weight at 1000 mg/kg/day, no toxicity was observed.

F1-generation, post-weaning: During the initial weeks the HD groups showed recovery of the slightly lower BW. No test item-related changes were noted.

F1-generation, neurotoxicity evaluation: No test item-related changes were noted.

Thyroid hormone analysis (T3, T4 and TSH) on post-natal day 21 and 70 showed no significant effects.

The study concluded to a NOAEL of at least 1000 mg/kg bw/day for both maternal and developmental toxicity.

 

As there are no consistent effects on thyroid hormones observed from the various studies that included serum hormone evaluations, i.e. in 90-day/repro screen (OECD 408/422), developmental neuro toxicity testing (OECD 426) and prenatal developmental toxicity testing in rat (OECD 414). Also in view of the absence of clear adverse effects in any of these studies, the overall NOAEL for repeated dose can be set at 1000 mg/kg bw.

Also no effects on reproduction has been observed up to 1000 mg/kg bw.

Effects on developmental toxicity

Description of key information

In a combined OECD 422/408 study at 1000 mg/kg bw/d a skewed sex ratio towards females, a reduced live birth index, increased postnatal loss and decreased body weights at 1000 mg/kg are observed which was possibly related to treatment. In a subsequent developmental toxicity in rats (OECD 414). This concluded to a developmental NOAEL of 300 mg/kg bw/day, based on the 6% lower fetal body weights observed at 1000 mg/kg bw/day when compared to concurrent controls as only effect, whereas 19% lower maternal lower body weight gain after correction for gravid uterus weight at 1000 mg/kg/day group compared to control was not considered adverse.An additional DNT study involving also dosing from implantation (gd 6) throughout the whole pregnancy and continuing during lactation, did not result to result to a significant difference in BW of the pups, although there was a slightly lower BW of the pups at 1000 mg/kg some weeks during lactation period, but post weaning there were no differences at all.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Qualifier:
according to guideline
Guideline:
other: EC No 440/2008, B.26 Sub-chronic Oral Toxicity Test: Repeated dose 90-day toxicity study in rodents
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
other: OECD 408. Repeated Dose 90-day Oral Toxicity Study in Rodents
Version / remarks:
1998
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3100, 90-Day Oral Toxicity in Rodents
Version / remarks:
1998
Principles of method if other than guideline:
In addition, the procedures described in this study plan essentially conform to the following guidelines:
OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Details on test animals or test system and environmental conditions:
Untreated, nulliparous, non-pregnant females and untreated males were used at the initiation of the study.

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- At initiation of dosing males and females were 6 weeks old, males weighed between 154 and 187 g and females weighed between 120 and 145 g.
- Fasting period before study: no
- Housing: Pretest and pre-mating: animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm); Mating: Males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm); Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm); Lactation: Females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) (During motor activity measurements, animals did not have access to food for a maximum of 2 hours)
- Water: Free access to tap-water (During motor activity measurements, animals did not have access to water for a maximum of 2 hours)
- Acclimation period: for 7 days prior to start of treatment;

DETAILS OF FOOD AND WATER QUALITY:
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
(set to maintain)
- Temperature (°C): 20 to 22
- Humidity (%): 43 to 66
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 April 2017 To: 02 August 2017
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
specific gravity 1.036
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily as solution (low and mid dose) or suspension (high dose) within 5 hours prior to dosing and were homogenized to a visually acceptable level. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of samples of formulations taken in week 1, 5 and 13 during the treatment phase were analysed according to a validated method. The samples were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were in agreement with the target concentrations (i.e. 90-110% for the low and mid dose and 85-115% for high dose). Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of formulations over 5 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study.
Details on mating procedure:
After 8 weeks of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which the females who had not shown evidence of mating were separated from her male.
Duration of treatment / exposure:
Males were treated for 92 days, i.e. 8 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were treated for 93-98 days, i.e. during 8 weeks prior to mating, the variable time to conception, the duration of the pregnancy and 14-15 days after delivery up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were treated for 85-96 days.
Routinely, females that are littering are left undisturbed. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
Frequency of treatment:
Once daily for 7 days per week
Duration of test:
See duration of treatment.
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:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Based on the results of a 14-day dose range finding study the dose levels for this combined 90-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg bw/day. In this DRF no mortality or no abnormalities were noted at 500 and 1000 mg/kg bw/day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
Ovaries and uterine content:
The ovaries and uterine content was examined after termination. Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
Sex: Sex was determined for all pups on PND 1 and 4. Sex ratio (% male pups / % female pups) was calculated per group.
Areola/Nipple retention: all male pups in each litter were examined for the numbe rof areola/nipples on PND 13.
Blood of F1-animals was collected on PND 4 and PND 13-15. Measurement of T4 was conducted for F0-animals and PND 13-15 pups. On account of the treatment-related changes in T4 in F0-males and thyroid morphology in F0-females (see respective results sections for details) assessment of thyroid hormone in parental animals was extended to T4 in females and TSH in both sexes.
All remaining pups were euthanized on PND 13-15. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or % CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Precoital time = Number of days between initiation of cohabitation and confirmation of mating
Fertility 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

Post-implantation survival index (%) = (Total number of offspring born/Total number of uterine implantation sites) x 100
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
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
Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Lactation index (%) = (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Salivation seen among animals treated with the test item was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality occurred in two incidences only; the premature death of male no. 26 (300 mg/kg) and female no. 80 (1000 mg/kg) was considered unrelated to treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of females were considered not to be affected by treatment. The slightly higher mean body weights noted in 1000 mg/kg females at most time points (up to 6%, not statistically significant) were considered to represent normal biological variation and, therefore, regarded as unrelated to treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption (before and after correction for body weight) was considered not to be affected by treatment.
For females at 1000 mg/kg a statistically significant decreased relative food consumption was noted on Days 4-7 of lactation. This was caused by the lower food consumption of female no. 75, which was related to her small litter size (i.e. one pup).
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment.
The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related changes in red and white blood cell parameters. The few statistically significant variations noted in females (higher mean values for total white blood cells, lymphocytes and red blood cell distribution width at 100 or 300 mg/kg) were considered unrelated to treatment due to the lack of a dose-related response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Lower ASAT levels in treated females (all dose groups) was related to the relatively high mean control value, due to an abnormal high values in two control females (no. 45 and 49).

Serum T4 levels in F0-males and F0-females were statistically significantly reduced (by 57% and 19%, respectively) at 1000 mg/kg. The statistically significantly lower (about 20%) mean T4 value noted in males at 100 mg/kg was considered unrelated to treatment due to the lack of a dose response (mean T4 at 300 mg/kg was similar to the control mean). Moreover, individual T4 values at 100 mg/kg generally remained within the concurrent and historical control range .
There were no treatment-related changes in serum TSH levels of both F0-males and F0-females. Overall, mean values of males and females at 100 and 300 mg/kg and males at 1000 mg/kg appeared higher than concurrent control mean values. Due to the relatively high standard deviations of these groups and a lack of a dose response, the higher mean values of the treated animals were considered as not toxicologically relevant. The statistically significantly higher (about four times higher than the control value) mean TSH value noted at 300 mg/kg was considered unrelated to treatment as the increase was caused by abnormal values in two single males (nos. 22 and 29).

Historical control data for T4 values in Wistar Han rats from OECD 422 studies
Males mean: 4.65, P5-P95: 2.970-6.440 (n=448; period 2015-2017)
Females mean: 3.57, P5-P95: 2.183-5.250 (n=40; period 2015-2017)
Note: males within historical control data were 14 weeks, whereas males within the current study were about 19 weeks old at time of blood sampling.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength was considered not to be affected by treatment. The statistically significant differences noted in hind limb grip strength (lower mean values in males at 100 and 1000 mg/kg and in females at 300 mg/kg) showed no clear dose-related response and were not accompanied by corroborative changes in other measures in the neuromuscular domain (including fore limb grip strength, gait, static righting reflex and motor activity). Moreover, the difference from controls was slight and all values remained within normal limits. Therefore, these findings were regarded as unrelated to treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Organ weights were considered not to be directly affected by treatment.
The statistically significant organ weight differences noted in 1000 mg/kg males (13% higher relative testes weight and 15% lower absolute prostate weight) were considered secondary to the (test item-related) lower body weights of these males (10% lower mean terminal body weight). Body weights of 300 mg/kg males were decreased to a similar degree but the changes in their testes and prostate weights (which were slightly smaller than those at 1000 mg/kg) were not statistically significant.
The other statistically significant variations noted in organ weight values for males (brain, thymus and adrenals in males at 100 and/or 300 mg/kg) were considered unrelated to treatment due to the lack of a dose-related response.
No treatment-related effects were observed in organ weights of females up to 1000 mg/kg.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations.
A slightly higher incidence of irregular surface of the stomach was noted in 3/10, 2/10, 4/10 and 1/9 females in the control, 100, 300 and 1000 mg/kg groups, respectively. This finding was considered unrelated to treatment due to the lack of a dose response.
All of the remaining recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the thyroid gland, an increased incidence of follicular cell hypertrophy was present in 1000 mg/kg females (up to slight).
In the urinary bladder, an increased incidence of hyperplasia/hypertrophy of the urothelium was present in 1000 mg/kg females (minimal). The urothelial change was characterized by a mixture of hyperplasia of the basal cell layer and/or hypertrophy of the dome-shaped surface cells (umbrella cells). The cells of the urothelium formed a uniform thickness (diffuse) without prominent focal outward or inward growth and without cellular atypia. There was no inflammation or cell death involved in this process.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A finding of note was observed in an ovary of 1000 mg/kg/day treated female (no.78) consisting of a tubular adenoma (also known as tubulostromal adenoma). Since there were no other test item-related proliferative findings observed in the ovaries, this incidental finding was considered as unrelated to the test item.
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) at 1000 mg/kg was relatively low compared to the concurrent control level (84% versus 89%). Also compared to historical control data, the calculated post-implantation survival index at 1000 mg/kg was at the lower limit of the normal range .
In the nine pregnant females treated at 1000 mg/kg, the individual difference between number of implantation sites and number of pups born (the so-called unaccounted for (implantation) sites) varied between 0 and 4, were 4/9 females had 3, 1/9 had 4 and 4/9 had 0-2 unaccounted for sites, respectively. For comparison, in the nine pregnant control group females 1/9 females had 3 and 8/9 females had 0-2 unaccounted for sites. These results indicated a trend of increased number of females with a higher number of unaccounted for sites, outside the range of that observed in concurrent controls, at 1000 mg/kg.
For female no. 70 (300 mg/kg) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) at 1000 mg/kg appeared lower than that at the other dose levels (9.8 versus 11.4, 12.2 and 11.2 at 0 (control), 100 and 300 mg/kg, respectively). The difference was not statistically significant and could largely be explained by the low number of live pups in a single litter (at first litter check, 1000 mg/kg female no. 75 had 2 live pups and 7 dead pups). Such a small live litter size occasionally occurs in untreated controls. All other litters at 1000 mg/kg had normal live litter sizes . Therefore, the apparently lower mean live litter size at 1000 mg/kg was considered not to reflect an adverse effect of the test item.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) at 1000 mg/kg (91%) was lower than that at the other dose levels (100, 100 and 99% at 0 (control), 100 and 300 mg/kg, respectively). At first litter check, one pup at 300 mg/kg (no. 66) and 9 pups at 1000 mg/kg (one of litter nos. 71 and 79, seven of litter no. 75) were found dead.

Viability indices (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment up to 300 mg/kg. The viability indices were 100, 98, 100 and 95% at 0 (control), 100, 300 and 1000 mg/kg, respectively.
One pup at 100 mg/kg (litter no. 52) and four pups at 1000 mg/kg (litter nos. 75, 78 and 79) went missing (presumably cannibalized) between PND 2-4. One pup at 300 mg/kg (litter no. 66) and nine pups at 1000 mg/kg (litter nos. 71, 75 and 79) were found dead on PND 1. In addition, one pup at 300 mg/kg was sacrificed early on PND 4, based on its poor physical condition (i.e. lean appearance). At 1000 mg/kg, the postnatal loss differed statistically significantly from that in the control group, in which no pups died. Also compared to historical control data the postnatal loss at 1000 mg/kg was higher .

Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.
Description (incidence and severity):
Gestation index and duration of gestation were not affected by treatment.
Description (incidence and severity):
Gestation index and duration of gestation were not affected by treatment. All pregnant females had live offspring (gestation index 100% for all groups).
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
maternal
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: thyroid
Description (incidence and severity):
decreased T4 levels in combination with increased incidence of follicular cell hypertrophy in the thryoid
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, mean pup weights on PND 1 were 9% (male pups) or 8% (female pups) lower compared to controls. Combined mean pup weights at 1000 mg/kg were statistically significantly lower, however remained within the normal range . Mean pup weights at 1000 mg/kg remained slightly lower at PND 4 and 7, but were close to control values at PND 13. A finding of note was the abnormally low birth weight and severely reduced postnatal growth of the single surviving female pup of 1000 mg/kg female no. 75. Based on the incidental occurrence, the reduced growth of this pup was regarded not toxicologically significant.
Pup weights at 100 and 300 mg/kg showed no remarkable differences from control weights.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) at 1000 mg/kg appeared lower than that at the other dose levels (9.8 versus 11.4, 12.2 and 11.2 at 0 (control), 100 and 300 mg/kg, respectively). The difference was not statistically significant and could largely be explained by the low number of live pups in a single litter (at first litter check, 1000 mg/kg female no. 75 had 2 live pups and 7 dead pups). Such a small live litter size occasionally occurs in untreated controls. All other litters at 1000 mg/kg had normal live litter sizes . Therefore, the apparently lower mean live litter size at 1000 mg/kg was considered not to reflect an adverse effect of the test item.

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) at 1000 mg/kg was relatively low compared to the concurrent control level (84% versus 89%). Also compared to historical control data, the calculated post-implantation survival index at 1000 mg/kg was at the lower limit of the normal range .
In the nine pregnant females treated at 1000 mg/kg, the individual difference between number of implantation sites and number of pups born (the so-called unaccounted for (implantation) sites) varied between 0 and 4, were 4/9 females had 3, 1/9 had 4 and 4/9 had 0-2 unaccounted for sites, respectively. For comparison, in the nine pregnant control group females 1/9 females had 3 and 8/9 females had 0-2 unaccounted for sites. These results indicated a trend of increased number of females with a higher number of unaccounted for sites, outside the range of that observed in concurrent controls, at 1000 mg/kg.
For female no. 70 (300 mg/kg) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) at 1000 mg/kg (91%) was lower than that at the other dose levels (100, 100 and 99% at 0 (control), 100 and 300 mg/kg, respectively). At first litter check, one pup at 300 mg/kg (no. 66) and 9 pups at 1000 mg/kg (one of litter nos. 71 and 79, seven of litter no. 75) were found dead.

Viability indices (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment up to 300 mg/kg. The viability indices were 100, 98, 100 and 95% at 0 (control), 100, 300 and 1000 mg/kg, respectively.
One pup at 100 mg/kg (litter no. 52) and four pups at 1000 mg/kg (litter nos. 75, 78 and 79) went missing (presumably cannibalized) between PND 2-4. One pup at 300 mg/kg (litter no. 66) and nine pups at 1000 mg/kg (litter nos. 71, 75 and 79) were found dead on PND 1. In addition, one pup at 300 mg/kg was sacrificed early on PND 4, based on its poor physical condition (i.e. lean appearance). At 1000 mg/kg, the postnatal loss differed statistically significantly from that in the control group, in which no pups died. Also compared to historical control data the postnatal loss at 1000 mg/kg was higher .

Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
The sex ratio of living pups at first litter check in the 1000 mg/kg group differed statistically significantly from that in the control group (% males/females: 34/66 at 1000 mg/kg versus 57/43 in the control group). On an individual litter basis, at 1000 mg/kg the percentage of female pups was about 70-80% in 5/8 litters of normal sizes and 100% in the litter that included only two live pups. For comparison, in the control group the percentage of female pups was about 80% in 1/9 litters and 55% or lower in the remaining litters. The sex ratios at 100 and 300 mg/kg were unremarkable (55/45 and 45/55, respectively).
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) at 1000 mg/kg appeared lower than that at the other dose levels (9.8 versus 11.4, 12.2 and 11.2 at 0 (control), 100 and 300 mg/kg, respectively). The difference was not statistically significant and could largely be explained by the low number of live pups in a single litter (at first litter check, 1000 mg/kg female no. 75 had 2 live pups and 7 dead pups). Such a small live litter size occasionally occurs in untreated controls. All other litters at 1000 mg/kg had normal live litter sizes . Therefore, the apparently lower mean live litter size at 1000 mg/kg was considered not to reflect an adverse effect of the test item.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
Viability indices (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment up to 300 mg/kg. The viability indices were 100, 98, 100 and 95% at 0 (control), 100, 300 and 1000 mg/kg, respectively.
One pup at 100 mg/kg (litter no. 52) and four pups at 1000 mg/kg (litter nos. 75, 78 and 79) went missing (presumably cannibalized) between PND 2-4. One pup at 300 mg/kg (litter no. 66) and nine pups at 1000 mg/kg (litter nos. 71, 75 and 79) were found dead on PND 1. In addition, one pup at 300 mg/kg was sacrificed early on PND 4, based on its poor physical condition (i.e. lean appearance). At 1000 mg/kg, the postnatal loss differed statistically significantly from that in the control group, in which no pups died. Also compared to historical control data the postnatal loss at 1000 mg/kg was higher .

Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the findings noted (i.e. alopecia) remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were considered unaffected by treatment.

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
A statistically significantly higher mean value for normalized anogenital distance in female pups at 1000 mg/kg was observed when compared to mean control value (0.59 vs. 0.53, respectively). As the mean values remained within historical control data , the difference between mean control and mean 1000 mg/kg values was minimal and due to the lack of a dose-related response, the increased mean value at 1000 mg/kg was not attributed to treatment.


Treatment up to 1000 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in postnatal survival
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
yes
Conclusions:
Based on decreased T4 levels (for females in combination with increased incidence of follicular cell hyperthrophy in the thyroid) at 1000 mg/kg bw/day, a NOAEL of 300 mg/kg bw/day is derived for parental toxicity.
No fertility effects were observed, therefore, the reproduction NOAEL is concluded to be at least 1000 mg/kg bw/day.
Based on a skewed sex ratio towards females, a reduced live birth index, increased postnatal loss and decreased body weights at 1000 mg/kg bw/day, a NOAEL of 300 mg/kg bw/day is derived for developmental toxicity.
Executive summary:

Wistar Han rats were treated with Ethylene glycol dibenzoate by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, polyethylene glycol 400, alone. Males were treated for 8 weeks prior to mating, during mating, and up to termination (for 92 days). Females that delivered offspring were treated for 8 weeks prior to mating, during mating, duringpost-coitum, and 14-15 days of lactation (for 93-98 days). Females without offspring were treated for 85 days (two non-pregnant females) or 96 days (one female without evidence of mating).

Formulation analysis showed that the formulations were prepared accurately and homogeneously.

Parental results

In females treated at 1000 mg/kg microscopic examination revealed an increased incidence of follicular cell hypertrophy (minimal or slight) in the thyroid. This was accompanied by a decrease of thyroid hormone T4 (on average 19%). In males treated at 1000 mg/kg serum levels of thyroid hormone T4 were also decreased (on average by 57%), unaccompanied by treatment related changes in thyroid weight or morphology. For both males and females no corroborative findings were observed in TSH levels. Based on the minimal or slight increase in follicular cell hypertrophy in combination with a decrease in T4 levels in 1000 mg/kg treated femalesand the magnitude of decreased T4 levels in 1000 mg/kg treated males, adversity could not be excluded.

Exposure to the test item up to 1000 mg/kg was not associated with obvious toxicity in the remaining parental parameters examined in this study (i.e. mortality, clinical appearance, functional tests, ophthalmoscopy, body weight, food consumption, clinical pathology, macroscopic examination and organ weights). Other changes noted in treated rats, mostly at 1000 mg/kg, were considered non-adverse as discussed below.

Slight salivation observed after dosing among treated animals (all dose levels) was considered a physiological response rather than a sign of systemic toxicity.

Male rats administered the test item showed reduced body weight gain from start treatment onwards, which was not accompanied by reduced food consumption. This occurred to a similar degree at 300 and 1000 mg/kg, and to a lesser degree at 100 mg/kg. The resulting reduction in mean body weights was modest (about 10% from Day 36 onwards at 300 and 1000 mg/kg) and the growth rate of treated males had returned to control levels after five weeks of treatment. Therefore, the decreased body weights of treated male rats were regarded as non-adverse within the context of this study.

A further treatment-related morphological change observed in females treated at 1000 mg/kg consisted of an increased incidence of minimal hyperplasia/hypertrophy of the urothelium of the urinary bladder. There was no inflammation or cell death involved in this process. Therefore, this minimal change was considered as non-adverse (Frazieret al., 20121).

Clinical pathology showed several changes in males treated at 1000 mg/kg: higher values for aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total bilirubin and albumin; and lower values for cholesterol. Additionally, the increased prothrombin time (PT) for males at 1000 mg/kg could be related to the decrease in the number of platelets. These changes were not accompanied by adverse anatomic pathology alterations and the mean values for these parameters in 1000 mg/kg males generally remained within the range considered normal for rats of this strain and age (see results section). As such, these clinical pathology changes were regarded as non-adverse (Palazziet al., 20162).

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs).

Developmental results

A skewed sex ratio towards females was noted at 1000 mg/kg: 66% female pups versus 43% in the control group (the difference was statistically significant due to reverse skewed sex-ratio in the control. Evaluations should have been for each group against historical control cq 50/50 expectation!). With such a high proportion of female pups it could not be excluded that the skewed sex ratio was related to treatment. This was strengthened by the finding that the percentage of female pups was about 70-80% in 5/8 litters of normal size at 1000 mg/kg versus 1/9 in the control group. There were no treatment-related changes in nipple retention in male pups, anogenital distances in male and female pups, or reproductive organs of parental animals. Therefore, it was considered unlikely that the skewed sex ratio resulted from an endocrine mediated (antiandrogenic) effect. An alternative explanation for the skewed sex ratio could be selective loss of male embryos due to a sex difference in sensitivity to toxic effects of the test item. The slightly increased number of unaccounted for implantation sites noted at 1000 mg/kg (resulting in a slightly lower live litter size) might reflect higher sensitivity of male embryos but does not constitute conclusive evidence of selective loss of male embryos. Comparison between ‘Total number of offspring born’ and ‘Total number of uterine implantation sites’ shows a rather comparable loss after implantation of 11.2% in control and 15.7% at 1000 mg/kg bw.

Live birth index at 1000 mg/kg was lower compared to controls (91% versus 100%). In total, 9 pups of the 1000 mg/kg group were found dead at first litter check: 7/9 of one litter (2 male and 5 female pups) and one male pup in two other litters. Additionally, a slight increase in postnatal loss was noted: 4 pups in three litters died between PND 1 and 4. Furthermore, decreased mean pup weights were noted at 1000 mg/kg on PND 1 (statistically significant for combined mean weights, relative to control 11% - however, as male and female pups differ in BW, the skewed sex-ratio with high proportion of females causes contributes to the difference. BW can only be compared for male and female pups separately.). Mean pup weights at 1000 mg/kg remained slightly lower at PND 4 and 7, but were close to control values at PND 13. The report concludes that based on the combination of a decreased live birth index, a slight increase in postnatal loss and decrease mean pup weights at PND 1, a treatment-related effect on the early viability of the pups could not be excluded. Attached are graphs of the development of the BW relative to the control during PND 1 to 13 for male respectively female pups. The 1000 mg/kg group shows a lower BW on PND 1 compared to the control, but as can be seen, it as rather similar for the 100 mg/kg group, whereas the 300 mg/kg is the same as control. The clear lack of a dose-response indicates that the observed difference is likely for a large part just fortuitous.

Further evaluation of number of corpora lutea and number of implantations

 

No treatment-related relevant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation index and duration, lactation indices, parturition, maternal care and the postnatal pup parameters clinical signs, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum concentration of thyroid hormone T4 (PND 13-15) and macroscopy).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Feb 2020 - 11 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han
Details on test animals or test system and environmental conditions:
The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 181 and 258 g
- Fasting period before study: no
- Housing: individually in Makrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and equipped with water bottles.
- Diet (ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (ad libitum): Municipal tap water
- Acclimation period: at least 5 days
- For psychological/environmental enrichment and nesting material, animals were provided with paper and aspen wooden sticks, except when interrupted by study procedures/activities.
- It was considered that there were no known contaminants in the feed or in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 46 to 52
- Air changes (per hr): Ten or greater, 100% fresh air, no air recirculation
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 16 Feb 2020 to 05 Mar 2020
Route of administration:
oral: gavage
Vehicle:
other: Polyethylene Glycol 400
Remarks:
Specific gravity: 1.125
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a suspension. They were heated to a maximum temperature of 60 ± 5°C for at least 30 minutes to obtain visual homogeneity. Formulations were filled out in daily portions and stored in the refrigerator until use. On the morning of administration, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 5 hours after removal from the refrigerator.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
The dose volume for each animal was based on the most recent body weight measurement. The dosing formulations were stirred continuously during dose administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected in week 1 of dosing. The acuracy of preparation (concentration) was analysed for all groups, homogeneity was checked for groups 2 and 4. The homogeneity results obtained from the top, middle and bottom for the preparations of Groups 2 and 4 were averaged and utilized as the concentration results.
Analyses were performed using a validated method. Concentration results were considered acceptable if mean sample concentration results were within or equal to ±15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was lower or equal to 10%.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 516416) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Details on mating procedure:
Females were already mated upon arrival.
Duration of treatment / exposure:
Day 6 to Day 20 post-coitum, inclusive
Frequency of treatment:
Once daily
Duration of test:
Animals were euthanized by an oxygen/carbon dioxide procedure on Day 21 post-coitum.
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
- Dose selection rationale: Dose levels were selected based on the results obtained in a Combined 90-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of Ethylene Glycol Dibenzoate by Oral Gavage in Rats (Test Facility Study No. 516410), and in an attempt to produce graded responses to the test item. In this study, dose levels of 100, 300 and 1000 mg/kg bw/day were administered by daily oral gavage. A parental NOAEL of 300 mg/kg bw/day was established based on decreased Thyroxine (T4) levels (for females in combination with increased incidence of follicular cell hypertrophy in the thyroid) at 1000 mg/kg bw/day. The NOAEL for reproductive toxicity was established at 1000 mg/kg bw/day, and the NOAEL for developmental toxicity was established at 300 mg/kg bw/day, based on a skewed sex ratio towards females, a reduced live birth index, increased postnatal loss and decreased body weights at 1000 mg/kg bw/day. Since the dosing period in this study (93-98 days for females that delivered) extensively exceeds the dosing period in this prenatal and developmental toxicity study (14 days), it was considered that 1000 mg/kg bw/day could be used as highest dose level.

- Rationale for animal assignment (if not random): n.a.
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: between 7:00 and 9:00 a.m.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: Yes
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21.
- All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).
- The thyroid gland and uterus were weighed at necropsy for all animals. Organ weight as a percent of body weight (using the body weight on Day 21 post-coitum) was calculated.
- In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
- The thyroid gland from all animals were examined histopathologically.
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 implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number and distribution of live and dead fetuses, sex of each fetus based on the anogenital distance
Blood sampling:
- Plasma: No
- Serum: Yes
- Volume collected: 0.1 mL (target volume)
- Other: Blood samples were processed for serum, and serum was analyzed for the thyroid hormones Triiodothyronine (T3), Thyroxine (T4), and Thyroid-Stimulating Hormone (TSH).
TSH and T4 analysis with the IMMULITE® 1000 method, T3 using LC-MS according to the bioanalytical method validated in Test Facility Study No. 20213516.
Fetal examinations:
Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: yes. The AGD was normalized to the cube root of the fetal body weight.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

Parametric:
Datasets with at least 3 groups (control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

Incidence:
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and postimplantation loss.
Indices:
- Body Weight Gains: Calculated against the body weight on Day 6 post-coitum.
- Corrected Body Weight Gains: Body weight on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
- Relative Food Consumption: Calculated against the body weight for scheduled intervals.
- Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum.

For each group, the following calculations were performed:
Preimplantation loss (%): ((number of corpora lutea - number of implantation sites) x 100) / number of corpora lutea

Postimplantation loss (%): ((number of implantation sites - number of live fetuses) x 100) / number of implantation sites

The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%): (number of viable fetuses affected/litter x 100) / number of viable fetuses/litter
Historical control data:
Historical control data on fetal examination were provided.
Test species: Rat (Crl:WI(Han) (ourbred, SPF Quality)),
Study range: 2015-2019
No. of studies: 60
Total No. of dams in the control group: 1321
Total No. of fetuses/Litters examined externally: 13756
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations did not reveal any alterations that were considered to have arisen as a result of dosing with the test item.
Salivation (slight) was seen after dosing in 5/22 females treated at 1000 mg/kg bw/day, mainly in the second half of the dosing period. This was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in body weight and body weight gain were noted following dosing up to 1000 mg/kg bw/day.
Mean body weights and body weight gains of treated animals remained in the same range as controls over the dosing period. However, after correction for gravid uterus weight mean body weight gain was slightly, but statistically significantly lower at 1000 mg/kg bw/day when compared to the concurrent control group (24.7 gram vs 30.4 gram; 0.81x of control). As the mean value of the high dose group remained well within the available historical control range , no toxicological relevance was attached to this finding.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was comparable to the control level following dosing up to 1000 mg/kg bw/day.
The slightly, but statistically significantly lower absolute food consumption observed in females treated at 100 mg/kg bw/day from Days 15-18 post-coitum and in females at 1000 mg/kg bw/day from Days 18-21 post-coitum was considered unrelated to dosing with the test item since no clear dose-related trend could be established.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Note: At 300 mg/kg bw/day, one female was not gravid and was therefore excluded from the data tables.
A trend towards slightly lower serum levels of thyroid stimulating hormone (TSH), total triiodothyronine (T3) and total thyroxine (T4) was observed from 100 mg/kg bw/day onwards, as is indicated in the table below. Changes compared to the concurrent control group were relatively small and did not always reached statistical significance. These changes were considered test item-related, but not adverse since all mean values remained within the available historical control range See table on thyroid hormone analysis in "Any other information on results incl. tables".
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Note: At 300 mg/kg bw/day, one female was not gravid and was therefore excluded from the data tables.
There were no treatment-related effects attributed to administration of the test item.
Higher thyroid gland weights (absolute and relative to body weight) were noted in the 100, 300 and 1000 mg/kg bw/day group females, compared to the concurrent control group (not statistically significant at 300 mg/kg bw/day). This was regarded to be unrelated to dosing with the test item, based on absence of a clear dose-response. Moreover, all means remained within the range considered normal for rats of this age and strain, and no macroscopic or microscopic correlate was present.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of dosing with the test item.
Incidental findings that were noted among control and/or treated animals included a control female with two missing toes, one female at 300 mg/kg bw/day with agenesis of thyroid gland and another female at this mid dose with many dark-red foci on the thyroid gland. These findings are occasionally seen among rats used in these types of study. At the isolated incidence and in absence of a dose-related trend they were considered unrelated to dosing with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations in the thyroid glands.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Litter incidence of pre implantation loss, as well as litter incidence of post-implantation loss in the control and test item groups were similar. All values remained within the normal range of biological variation.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
Mean numbers of early and late resorptions in the control and test item groups were similar. All values remained within the normal range of biological variation.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Numbers of pregnant females and mean numbers of corpora lutea in the control and test item groups were similar. All values remained within the normal range of biological variation.

At 300 mg/kg bw/day, one female was not pregnant, i.e. she had no corpora lutea and no implantation sites (confirmed by Salewski staining; taken from raw data). This isolated case of non-pregnancy was unrelated to dosing with the test item as it occurred at the mid dose only.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: The for uterus corrected maternal body weight gain decrease of 19% when compared to concurrent controls was not considered adverse.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male and female fetal body weights were significantly reduced at 1000 mg/kg bw/day (relative difference to concurrent controls: -6% for both, males and females). Values were below the lower limit of the historical control range.
Fetal weights (male, female and combined) in the 100 and 300 mg/kg bw/day dose groups were comparable to the concurrent control group and remained within the historical control range of the Test Facility.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
In the 1000 mg/kg bw/day group, mean percentage of viable fetuses per litter was statistically significantly increased when compared to the concurrent control group. Mean incidences were 90.2, 93.4, 95.3 and 98.8% in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. This was accompanied by a decreased mean litter incidence of early resorptions, total resorptions and post-implantation loss in the high dose group. Mean values were just below the available range of historical control data. However, in view of the direction of change these observations were considered unlikely to be related to dosing with the test item.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no changes in litter size observed that were considered to be related to dosing with the test item. No information on litter weight is available.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant effects on fetal anogenital distance (both sexes) noted after dosing up to 1000 mg/kg bw/day.
The statistically significantly higher mean value of corrected anogenital distance observed in male pups of the 1000 mg/kg bw/day group when compared to the concurrent controls was considered due to biological variation and does not signify a biological relevant change. The lower body weight of pups in this dose group aided to the relative increase in corrected anogenital distance. Mean value (1.54 mm) of the high dose group (1000 mg/kg bw/day) was identical to that of the low dose group (100 mg/kg bw/day), and all individual levels in high dose males (ranging from 1.27-1.67 mm) remained below the highest value (1.71 mm) measured in concurrent control males.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in external morphology noted that could be attributed to administration of the test item.
Four fetuses were externally malformed, two each in the 100 and 300 mg/kg bw/day groups. At 300 mg/kg bw/day, two littermates were affected. One of these 300 mg/kg bw/day fetuses had exencephaly and a small lower jaw, and at visceral examination, it was noted that also both eyes were absent. Skeletal examination substantiated these malformations and additionally revealed a vertebral and skull anomaly. The littermate at 300 mg/kg bw/day had a cleft palate and one missing eye bulge that both were confirmed at skeletal examination, whereby also a vertebral and rib anomaly was observed.
One of the two affected fetuses at 100 mg/kg bw/day had besides a small lower jaw and protruding tongue with matching skeletal jaw findings also a skull anomaly. The other fetus had an omphalocele.
These malformations were considered to be spontaneous in origin due to random occurrences and the lack of a dose response. Moreover, all findings except protruding tongue had been observed in historical control fetuses before.
No external variations were seen in any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in skeletal morphology noted that were considered to be a direct effect of exposure to the test item.
Besides the underlying skeletal malformations in the externally malformed fetuses at 100 and 300 mg/kg bw/day and their vertebral, rib and/or skull anomalies described in a paragraph above, a vertebral anomaly occurred in one fetus at 1000 mg/kg bw/day. All these findings are previously observed in historical control fetuses, and at the single or low incidences they occurred, they were considered chance findings.
At 1000 mg/kg bw/day, two fetuses had bent limb bones which also occurred in a dead fetus at 100 mg/kg bw/day and control fetus. The remaining malformations in this study, sternoschisis and vertebral centra anomaly occurred singly in a 300 mg/kg bw/day fetus and a control fetus, respectively. The incidence and group distribution of these findings does not indicate a relationship to treatment with the test item, and as all were listed in historical control data, they were considered chance findings.
Skeletal variations occurred at a mean litter incidence of 87.3%, 82.1%, 69.9% and 91.0% in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The incidence at 300 mg/kg bw/day was statistically significantly decreased compared to the control value, which was caused mainly by significant lower incidences of reduced ossification of the skull and bent ribs. Mean litter incidences for these respective variations were 28.1%, 22.4%, 7.8%, 35.1% and 35.1%, 15.0%, 13.5%, 39.8% in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The cause of less fetuses having reduced ossification of the skull and bent ribs at 300 mg/kg bw/day is not known, but considered not related to dosing with the test item due to absence of a dose related trend. Moreover, it was noted that the 300 mg/kg bw/day incidences for these two variations were within the historical control data range, while both the control and high dose incidences were above the maximum value.
Among the variations, the incidence of unossified metacarpals and/or metatarsals was increased at 1000 mg/kg bw/day. Incidences were 1.6%, 0.9%, 2.8% and 11.0% per litter in the control 100, 300 and 1000 mg/kg bw/day groups, respectively. The increase at 1000 mg/kg bw/day was not statistically significant and the value remained within the historical control data range (0.0% - 17.6% per litter). However, this parameter is a major indicator of delayed skeletal ossification, and all six affected male fetuses and 6/9 affected female fetuses had weights below the male or female group mean weights. Moreover, as group mean fetal weights at 1000 mg/kg bw/day were statistically significantly lower compared to the control value (4.9 versus 5.3 grams, respectively), it was considered that the higher incidence of unossified metacarpals and/or metatarsals at 1000 mg/kg bw/day was secondary to the fetal weight effect and not a direct effect of the test item.
The other variations occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within or near the range of available historical control data. Therefore, they were not considered related to dosing with the test item.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in visceral morphology noted that could be attributed to administration of the test item.
Besides the externally malformed fetus without eyes at 300 mg/kg bw/day described in the previous section, two cases of situs inversus were noted. These occurred in a 300 mg/kg bw/day fetus and a control fetus which did not indicate a relationship to dosing with the test item.
The visceral variations that were noted occurred infrequently, in the absence of a dose-related incidence trend, in control fetuses exclusively and/or at frequencies that were within the range of available historical control data.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
No changes in external, skeletal or visceral morphology noted that could be attributed to administration of the test item.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Dose formulation analysis

Accuracy

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks. The maximal contribution to the Group 2 sample was as low as 0.0028%, and therefore considered negligible.

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

 

Thyroid Hormone analysis

Mean Percent Differences in Thyroid Hormone Levels from Control Group

 

Dose level (mg/kg/day)

100

300

1000

TSH

-4

-15

-19

Total T3

-8

-15*

-24**

Total T4

-10

-13

-18*

*: P<0.05, **: P<0.01

 

Historical Control Data

Historical control data on maternal developmental effects such as pregnacy, corpora lutea, implanation loss, and on fetal parameters such as viable fetuses, sex, weight, AGD, malformations and variations can be found in appendix 3 of the study report.

Conclusions:
Maternal NOAEL:   1000 mg/kg bw/day (the for uterus corrected maternal body weight gain decrease of 19% when compared to concurrent controls was not considered adverse).
Developmental NOAEL: 300 mg/kg bw/day (based on the 6% lower fetal body weights observed at 1000 mg/kg bw/day when compared to concurrent controls).
Executive summary:

The objectives of this study were to determine the potential of Ethylene Glycol Dibenzoate (EGDB) to induce developmental toxicity in the rat after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested.

Time-mated female Wistar Han rats (22 females/dose level) were treated with Ethylene Glycol Dibenzoate by daily oral gavage from Day 6 to Day 20 post-coitum, inclusive, at 0, 100, 300, 1000 mg/kg bw/day. The study is performed in accordance with OECD 414 (June 2018), EU Method B.31 (2008) and EPA OPPTS 870.3700 (1998).

Formulation analyses confirmed that the formulations were prepared accurately and homogenously.

 

No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

A trend towards slightly lower serum levels of thyroid stimulating hormone (TSH), and total triiodothyronine (T3) and thyroxine (T4) was observed from 100 mg/kg bw/day onwards. Changes compared to the concurrent control group were relatively small and did not always reach statistical significance. At the highest dose of 1000 mg/kg bw/day, mean values for TSH, T3 and T4 reached respectively 0.81x (not statistically significant), 0.76x and 0.82x of control. These changes were considered possibly test item-related, but not adverse since all mean values remained within the range of historical control data. This study is not suited to evaluate the possible mechanism how the observed effects on the hormones could be brought about, but it is noteworthy that the same direction and magnitude of the observed changes in the TSH level and the T3 and T4 levels, is against the normal physiological feedback mechanism for these hormones. (Attached graph shows that the small differences between averages of the groups for each of the hormones are insignificant compared to the individual variability.)

No test item-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, body weight, food consumption, gross pathology, organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and postimplantation loss). Although there was a trend of lower body weight gain after correction for gravid uterus weight, which was 19% lower for the 1000 mg/kg bw/day group compared to control (See attached graph), this was not considered to be adverse, based on available historical control.

 

No developmental toxicity was observed in the 100 and 300 mg/kg bw/day groups.

At 1000 mg/kg bw/day, mean male and female fetal body weights were statistically significant reduced (relative difference to concurrent controls: -6% for both sexes). As fetal body weights have a small variation in general and mean values at 1000 mg/kg bw/day were below the lower limit of the historical control range, these findings were considered adverse.

There was an increased litter incidence of unossified metacarpals and/or metatarsals observed. Incidences were 1.6%, 0.9%, 2.8% and 11.0% per litter in the control 100, 300 and 1000 mg/kg bw/day groups, respectively. The increase at 1000 mg/kg bw/day was not statistically significant and the value remained within the historical control data range (0.0% - 17.6% per litter).

Both the lower pup BW, and the increased litter incidence of unossified metacarpals and/or metatarsals were minimal and hardly significant, as was the 19% lower body weight gain after correction for gravid uterus weight in the dams all at 1000 mg/kg bw. Together it is suggestive for a possible secondary effect of delayed fetal growth, and not a direct effect of the test item.

Remarkable, but likely just to be fortuitous, are the a highly statistically significant increase in number of viable fetuses observed at 1000 mg/kg, as well as decreased early and total resorptions, and a decreased post-implantation loss. Otherwise, no test item-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, anogenital distance, external, visceral and skeletal malformations, and external and visceral developmental variations).

Based on the results in this prenatal developmental toxicity study with Ethylene Glycol Dibenzoate the maternal NOAEL is 1000 mg/kg bw/day and the developmental NOAEL is 300 mg/kg bw/day (based on the 6% lower fetal body weights observed at 1000 mg/kg bw/day when compared to concurrent controls).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A GLP and OECD guideline K1 study is available.
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

OECD 422:

Combined repeated dose (90-day) and reproduction toxicity screening (OECD 422):

10 rats/sex/group received 0, 100, 300 and 1000 mg/kg Ethylene glycol dibenzoate (EGDB) by daily oral gavage for 8 weeks prior to mating, during mating, and up to termination (for 92 days males) after 14-15 days of lactation (for 93-98 days females).

Parental results:

Based on decreased T4 levels (for females in combination with increased incidence of follicular cell hypertrophy in the thyroid) at 1000 mg/kg, of which adversity could not be excluded, a NOAEL of 300 mg/kg bw/day was derived for parental toxicity. All other parameters were without significant findings.

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs).

Developmental results

A skewed sex ratio towards females was noted at 1000 mg/kg: 66% female pups versus 43% in the control group (the difference was statistically significant due to reverse skewed sex-ratio in the control. Evaluations should have been for each group against historical control cq 50/50 expectation!). There were no treatment-related changes in nipple retention in male pups, anogenital distances in male and female pups, or reproductive organs of parental animals. The slightly increased number of unaccounted for implantation sites noted at 1000 mg/kg (resulting in a slightly lower live litter size) might reflect higher sensitivity of male embryos but does not constitute conclusive evidence of selective loss of male embryos. 

Live birth index at 1000 mg/kg was lower compared to controls (91% versus 100%). In total, 9 pups of the 1000 mg/kg group were found dead at first litter check: 7/9 of one litter (2 male and 5 female pups) and one male pup in two other litters. Additionally, a slight increase in postnatal loss was noted: 4 pups in three litters died between PND 1 and 4. Furthermore, decreased mean pup weights were noted at 1000 mg/kg on PND 1 (statistically significant for combined mean weights, relative to control 11% - however, as male and female pups differ in BW, the skewed sex-ratio with high proportion of females causes contributes to the difference. BW can only be compared for male and female pups separately!). Mean pup weights at 1000 mg/kg remained slightly lower at PND 4 and 7, but were close to control values at PND 13. The report concludes that based on the combination of a decreased live birth index, a slight increase in postnatal loss and decrease mean pup weights at PND 1, a treatment-related effect on the early viability of the pups could not be excluded. Indeed, the 1000 mg/kg group shows a lower BW on PND 1 compared to the control, but as can be seen, it as rather similar for the 100 mg/kg group, whereas the 300 mg/kg is again the same as control. The clear lack of a dose-response indicates that the observed difference is likely for a large part just fortuitous.

No treatment-related relevant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation index and duration, lactation indices, parturition, maternal care and the postnatal pup parameters clinical signs, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum concentration of thyroid hormone T4 (PND 13-15) and macroscopy).

 

 

Further studies have been performed in order to evaluate the possible significance of the findings from this OECD 422 reproduction screening study:

 

OECD 414:

EGDB was tested for developmental toxicity in rats (OECD 414). 22 female rats were dosed at 0, 100, 300 and 1000 mg EGDB/kg bw in PEG 400 at 5 mL/kg bw/d in 22 female rats per dose group during gd 6 to 20.

Maternal results:

No maternal toxicity was observed. There was a trend of lower body weight gain after correction for gravid uterus weight, which was 19% lower for the 1000 mg/kg/day group compared to control, this was not considered to be adverse, based on available historical control data.

Thyroid related results:

- All thyroid hormones (TSH, T3, T4) seem to show a dose-response decrease. The decrease is relatively small, and values remain within historical control. Besides, the same trend for TSH and for T3&T4 signifies no biological impact.

- Thyroid histopathology showed no effects

- Thyroid weight is higher in dose groups compared to control. This was regarded to be unrelated to treatment with the test item, based on absence of a dose response, the fact that the values were within historical control data and the absence of a macroscopic or microscopic correlate. Most probably this just reflects accidentally somewhat lower average thyroid weights in the control group.

 

No developmental toxicity was observed in the 100 and 300 mg/kg/day groups. 

At 1000 mg/kg bw,a 6% lower fetal body weights observed at 1000 mg/kg/day when compared to concurrent controls), accompanied by increased litter incidence of unossified metacarpals and/or metatarsals.Incidences were 1.6%, 0.9%, 2.8% and 11.0% per litter in the control 100, 300 and 1000 mg/kg bw/day groups, respectively. The increase at 1000 mg/kg bw/day was not statistically significant and the value remained within the historical control data range (0.0% - 17.6% per litter).

Both the lower pup BW, and the increased litter incidence of unossified metacarpals and/or metatarsals were minimal and hardly significant, as was the 19% lower body weight gain after correction for gravid uterus weight in the dams all at 1000 mg/kg bw. Together it is suggestive for a possible secondary effect of delayed fetal growth, and not a direct effect of the test item.

- Remarkable, but likely just to be fortuitous are the a highly statistically significant increase in number of viable fetuses observed at 1000 mg/kg, as well as decreased early and total resorptions, and a decreased post-implantation loss

 

DNT:

In order assess the possible hazard for human thyroid insufficiency in adults as well as pre- and post-natal neurological development of offspring, a developmental neurotoxicity study (OECD 426) has been performed. Female rats were dosed at 0, 100, 300 and 1000 mg EGDB/kg bw in 25 female rats per dose group from gd 6 through to lactation day 20 (at weaning of the pups). Pups were not dosed directly; excretion into maternal milk from lactating dams was demonstrated in a lactational transfer study.

On PND 4 pups were culled to 8 per litter, and assigned 1 pup/sex/litter to 4 sub-sets (A,B,C and D) to make up 20/sex/grp for each of the four sub-sets.

Included were observations to detect gross neurologic and behavioural abnormalities, including the assessment of physical development, behavioural ontogeny, motor activity, motor and sensory function, and learning and memory and the evaluation of brain weights and neuropathology during postnatal development and adulthood.

Results:

F0-generation: Besides liver enlargement observed in 2 HD and 1 MD female, which was considered to be non-adverse, no maternal toxicity was observed. There were no effects observed on reproduction parameters.

F1-generation, pre-weaning: A non-adverse slightly lower mean pre-weaning body weight at 1000 mg/kg/day, no toxicity was observed.

F1-generation, post-weaning: During the initial weeks the HD groups showed recovery of the slightly lower BW. No test item-related changes were noted.

F1-generation, neurotoxicity evaluation: No test item-related changes were noted.

Thyroid hormone analysis (T3, T4 and TSH) on post-natal day 21 and 70 showed no significant effects.

The study concluded to a NOAEL of at least 1000 mg/kg bw/day for both maternal and developmental toxicity.

 

 

 

Overall evaluation:

The following concerns have been noted from the combined OECD 422/408 study, based on finding observed at the highest dose of 1000 mg/kg bw/day:

- Possible effects on thyroid:

This is based on decreased T4 levels (for females in combination with increased incidence of follicular cell hypertrophy in the thyroid) at 1000 mg/kg. Although the effects were not consistent, unaccompanied by treatment related changes in thyroid weight or morphology, and no corroborative findings were observed in TSH levels. The subsequent developmental toxicity in rat (OECD 414) and DNT study (developmental neurotoxicity OECD 426) did not show effects on thyroid hormones, nor on thyroid weights. The small increased incidence of minimal to slight follicular cell hypertrophy in the thyroid compared to control was only seen in the females at 1000 mg/kg, but not in the males although the suggested effects on thyroid hormones in males was much larger. As minimal to slight follicular cell hypertrophy in the thyroid as such also occurs in the control, the effects are not considered adverse. Based on structure and metabolism, EGDB can be expected to be comparable to DEGDB and Benzoic acid (see Mode of Action analysis). All three show a NOAEL of 1000 mg/kg bw/day from repeated dose toxicity testing, and for neither substance shows effects on thyroid.

- Skewed sex-ratio:

The OECD 422 study showed a shifted sex-ratio at 1000 mg/kg towards females (66% female pups). This was shown statistically significantly different when compared to the control showing a shift towards males (43% females). The statistical evaluation should however be against 50/50 and historical control. As further OECD 414 and DNT study did no show a skewed distribution, this means that if the effect of the shift is genuine test substance related, this must have been occurred as result either on sperm or on conception. Also considering that such effect is not seen for comparable substances Benzoic acid and DEGDB (See Mode of Action analysis), it is most likely that the skewed sex-ratio observed in the OECD 422 study is just a fortuitous finding.

-Reduced live birth index, increased postnatal loss:

De results from the OECD 422 study seems to indicate a slightly lower live birth index compared to control, and a small high postnatal loss of pups. These effects have not been confirmed in both OECD 414 and DNT studies, both involving dosing of the dams from implantation until end of study (gd 21 and lactation day 21 respectively). Actually, there was ahighly statistically significant increase in number of viable fetuses observed at 1000 mg/kg in the OECD 414, as well as decreased early and total resorptions, and a decreased post-implantation loss.

Most likely therefore are these observations just accidental.

- Reduced pup weight at birth:

This seems to be the only consistent finding over all studies. In attached document the mean BW relative to control is given of pups around birth (day 1 in OECD 422 and OECD 426, and on gd21 in OECD 414). This indicates a possible 3% to 6% lower BW of the pups compared to control for at birth at 1000 mg/kg dose level. In the OECD 414, the lower pup BW at 1000 mg/kg was accompanied by a minimal and hardly significant increased litter incidence of unossified metacarpals and/or metatarsals. These effects are minimal, and both the OECD 422 and the DNT studies show a complete recovery to same levels as control. In the OECD 414 study, the slightly lower BW of the pups is accompanied by a 19% lower body weight gain after correction for gravid uterus weight in the dams all at 1000 mg/kg bw.

 

In conclusion:

There on overall indication of a minimal lower BW of pups at birth, that was accompanied by a minimal and hardly significant increased litter incidence of unossified metacarpals and/or metatarsals at the highest dose of 1000 mg/kg bw. The OECD 422 and DNT studies showed that effects on BW quickly disappears, and no effects are seen at further development.

Mode of Action Analysis / Human Relevance Framework

Ethylene glycol dibenzoate (EGDB) is expected to be metabolized relative quickly over the two ester bonds in analogy of the comparable substance DEGDB (Oxydiethylene dibenzoate, CAS 120-55-8).In an OECD 417 study, virtually all DEGDB was absorbed, metabolized and excreted in urine with 24 hours of administration. Metabolism involved hydrolysis of the ester bonds to benzoic acid, which was conjugated with either glycine (major pathway) or glucuronic acid (minor pathway) prior to excretion.The same can be expected for EGDB.All three substances, benzoic acid, EGDB and DEGDB show a NOAEL of 1000 mg/kg bw/day from repeated dose toxicity testing, and neither substance have shown to be reproduction toxic.

Justification for classification or non-classification

The only effects seen at highest dose of 1000 mg/kg is a small, and not always statistically significant lower BW of pups at birth, which was accompanied by a minimal and hardly significant increased litter incidence of unossified metacarpals and/or metatarsals. Otherwise no effects on fertility, development and general toxicity observed. Also based on possible read-across to structurally comparable substances and main metabolite benzoic acid, there are no specific concerns from developmental toxicity.

Additional information