Reaction products of 2-hydroxyethyl methacrylate and diphosphorous pentoxide and water

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 26, 2016 to March 24, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 26, 2016 to March 24, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

but considered not to adversely affect the results or integrity of the study.

Qualifier:

according to guideline

Guideline:

other: OECD guidance document 43

Version / remarks:

The guideline was designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.

GLP compliance:

yes

Limit test:

no

Justification for study design:

- To obtain information on the possible toxic effects of the test substance following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels. A control group received the vehicle only (propylene glycol).
- The study included a reproductive/ developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance including gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 4 post-partum.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany)
Housing conditions: SPF at the supplier, standard laboratory conditions during the study
Number of animals: 51 male, 51 female rats, 4 groups. 12 animals/sex/group, with the exception of the High dose group, where 15 animals/sex/group was used. Animals originated from different units, to avoid brother/sister matings
Age of animals: young adult rats, at least 10 weeks old at starting and 12 weeks at mating
Body weight at the start: males: 362-422 g, females: 218-261 g
Acclimation period: at least 5 days
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.2-24.3 °C (target: 22 ± 3°C)
Relative humidity: 40 - 62% (target: 30 - 70%)
Ventilation: 15-20 air exchanges/hour
Food and water supply: ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" and tap water, ad libitum

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

The oral route was selected, as it is a possible route of exposure to the test substance in humans.

Details on mating procedure:

Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male from the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 14 days. If the first 14 days of the mating was unsuccessful, then the female was paired for a second period with a proven male from the same dose group (1:1 mating).
A vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

Analysis of test substance formulations for concentration and homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken and analysed from test substance formulations and all concentrations. Sample analysis was performed on 3 occasions (with an additional occasion for the High dose (250 mg/ml) formulation) for homogeneity (top, mid and bottom) and all prepared formulations were analysed for concentration. One set was collected for analysis and one set as a back-up. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of test substance. Description of the analytical method and the results of the formulation analysis are included in the Analytical report provided by the Analytical Laboratory of CiToxLAB Hungary Ltd.
Analysis of the test substance formulation samples of 1-250 mg/mL concentration range showed no decrease of concentration and can be considered as stable for 7 days at room temperature.
The measured concentrations of the test substance evaluated for each test substance-dose group varied between 94 % and 107 % of the nominal contents. No test substance was detected in the control samples. These results were within the acceptable range (85% - 115%) and are acceptable for the study purposes. All samples were found to be homogeneous.

Duration of treatment / exposure:

- Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating), and then euthanized and subjected to necropsy examination.
- Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. (Females showing no-evidence of copulation were sacrificed 25 days after the end of the mating period (two animals in the Control and two animals in the Mid dose, with no delivery).
- All F1 offspring were terminated on Day 4 post-partum. In order to allow for overnight fasting of dams prior to urine collection on PPD5, the offspring were euthanized on PPD/PND 4 and the dams on PPD/PND 5.

Frequency of treatment:

daily on a 7 days/week basis by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 4 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on most recent individual body weights. (Dosing of both sexes began after at least 5 days of acclimation and 2 weeks before mating and continued up to the day before necropsy).

Dose / conc.:

0 mg/kg bw/day

Remarks:

Concentration: 0 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

100 mg/kg bw/day

Remarks:

Concentration: 25 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

300 mg/kg bw/day

Remarks:

Concentration: 75 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Concentration: 250 mg/mL
Dose volume: 4 mL/kg bw

No. of animals per sex per dose:

at least 12

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work including the results of a repeated dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 15/340-220PE). The aim was to induce toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
Based on the results from these preliminary studies, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study.

Parental animals: Observations and examinations:

Refer to the study under repeated dose endpoint (section 7.5.1) for details on systemic parameter examinations.

Litter observations:

Observation of the delivery process, offspring and nursing instinct:
Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. Any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition. Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded. Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities. Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0 or PND1) and on PND4, with accuracy of 0.01g. All litters were checked daily for the number of viable and dead pups. All pups were sacrified on PND4.

Postmortem examinations (parental animals):

- Macroscopic evaluation
Gross necropsy was performed on all animals, irrespective of the date of death. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded in the females as applicable.

- Organ weight measurements

At the time of termination, body weight and the weight of the following organs from all euthanized adult animals were determined:
- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Paired organs were weighed together except testes and epididymides, which were weighed individually. Individual and/or paired absolute organ weights were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.

- Tissue preservation and microscopic evaluation
The weighed organs and all organs showing macroscopic lesions were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution; all other organs in 10% buffered formalin solution. The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Detailed histological examination was performed on all retained organs in the control and Hhigh dose groups and any macroscopic findings (abnormalities) observed in all animals. Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Postmortem examinations (offspring):

Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Statistics:

Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, or using the software PROVANTIS v.9, as appropriate. Group means and standard deviations were calculated from numerical data obtained in the study.
The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) by an appropriate statistical method (Bartlett, ANOVA/ANCOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests, T-test, Wilcoxon test, Chi2 test). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA/ANCOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test or Kruskal-Wallis test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible. For SMART evaluation, T-test was used.

Reproductive indices:

Male Mating Index % (Measure of male’s ability to mate): "Number of males with confirmed mating" /"Total number of males cohabited" "x100"

Female Mating Index % (Measure of female’s ability to mate): "Number of sperm-positive females" /"Total number of females cohabited" "x100"

Male Fertility Index % (Measure of male’s ability to produce sperm that can fertilise eggs): "Number of males impregnating a female" /"Total number of males cohabited" "x100"

Female Fertility Index % (Measure of female’s ability to become pregnant): "Number of pregnant females" /"Number of sperm-positive females" "x100"

Gestation Index % (Measure of pregnancy that provides at least one live pup): "Number of females with live born pups" /"Number of pregnant females" "x100"

Offspring viability indices:

Survival Index %: "Number of live pups (at designated time)" /"Number of pups born" "x100"

Pre-implantation mortality %: "Number of corpora lutea-Number of implantations" /"Number of corpora lutea" "x100"

Intrauterine mortality %: "Number of implantations-Number of liveborns" /"Number of implantations" "x100"

Total mortality %: "Number of implantations-Number of viable pups (PND4)" /"Number of implantations" "x100"

Sex ratio %: "Number of pups examined-Number of males" /"Number of pups examined" "x100"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
Terminally euthanised animals:
- Male animals: no clinical signs were detected in the Control, Low and Mid dose groups during the study. In the High (1000 mg/kg bw/day) dose group liquid faeces, noisy respiration and piloerection were occasionally present up to three animals during the treatment. These observations were considered to be treatment related.
- Female animals: thin fur/alopecia was observed in all groups, peaking at the end of the experiment, but is not considered to be related to test substance. No clearly adverse effects of the test substance related were noted in the Low dose groups during the study. In the Mid (300 mg/kg bw/day) dose group, piloerection and noisy respiration was observed sporadically in just 1 or 2 females. These non-specific observations seen sporadically at a low incidence is not considered to be a clear sign of an adverse effect of the test substance. In the High (1000 mg/kg bw/day) dose group, the non-specific signs of piloerection and noisy respiration was seen in a small number of male and the majority of females. Hunched back was also present in up to three female animals, which may be treatment related. A 1 to 2 cm wide crust was seen on the skin for five days in one animal, yellow coloured discharge in one animal, and discharge from vagina in one animal was observed during the treatment; these findings are of uncertain relationship with treatment.

Found dead and preterminally euthanized animals:
No common symptoms were found prior unanticipated deaths. The male animal showed slightly noisy, moderately laboured respiration one day before it was found dead. Two females showed no clinical signs before the time of death. Piloerection and slight to moderate noisy respiration and/or laboured respiration with hunched back was present in the rest of the animals, with red coloured discharge or distended abdomen prior dead or euthanasia. These symptoms were considered to be test substance related.

Excluded animals
Four females (two from the Control Dose group and two from the Mid (300 mg/kg bw/day) Dose group were excluded from certain statistical analysis (such as body weight) after being found to be sperm positive, but delivered no offspring despite detectable corpora lutea. Since body weight is affected by pregnancy, it is valid to exclude the non-pregnant animals from this data analysis. Sporadic occurrence of noisy respiration in one Control group and the two Mid dose animals, laboured respiration and piloerection in one Mid dose animal was observed, but these observations were not considered to be test item related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
A test substance related mortality (approximately 15% mortality) was observed in the High dose group (1000 mg/kg bw/day). One male (Day 16) and 4 females (Days 3, 6, 18 and 22 ) of the High dose group were found dead. In addition, one female (Day 17) was preterminally euthanized.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance related effects were noted on the mean body weight and body weight gain values following daily administration of the test substance at dose levels up to and including 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance related differences in the mean daily food consumption in any test substance treated group when compared to the controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
When compared to the controls, there were no differences that could be considered toxicologically significant in the Low and Mid dose (100 and 300 mg/kg bw/day, respectively) groups. The High (1000 mg/kg bw/day) males only had a slightly but significantly (p<0.05) lower Mean Cell Haemoglobin Concentratin (MCHC). The female High dose group had a slightly but significantly (p<0.05) longer Activated Partial Thromboplastin Time (APTT). However the data from all animals was in the normal range and these small differences were not consistent between sexes and were not considered to be an adverse effect of treatment.
For other parameters, evaluation of the mean and individual results in comparison with the control data did not reveal any test-substance related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range and of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
In the animals evaluated at termination (on Day 28 in males and on PPD5 in females), there were no clearly toxicologically significant changes or adverse effects on the animal serum chemistry that could be ascribed to the test substance administration in the conditions of this study. Urea and chloride concentrations were statistically significantly higher in the male High (1000 mg/kg bw/day) dose group compared to the Control (p<0.05 and p<0.01, respectively). The female High dose group had statistically significantly (p<0.05) lower phosphorus values. However the data for these 3 parameters from all animals was in the normal range and these small differences were not consistent between sexes and were not considered to be an adverse effect of treatment.
Some differences in the Low and Mid treated groups also attained statistical significance; however, there was no dose or gender response or the values were within the physiological ranges. For this reason, these variations were not considered toxicologically significant or related to treatment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
A dose dependent decrease of pH was observed in both sexes, reaching statistical significant difference compared to the control value at the High (1000 mg/kg bw/day) dose males (p<0.05) and females (p<0.01). In the High dose only, slight but statistical significance differences were recorded with lower pH and higher urine gravity in both sexes (p<0.01) and lower urine volume in makes only (p<0.05). However, all values were in the normal control range, these findings were regarded as minor variations and were not considered to indicate an adverse effect of the test substance.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
Neurological:
There were no toxicologically significant changes in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test groups.
There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. Grip strengths of the forelimb were significantly (p <0.05) lower in Mid dose (300 mg/kg bw/day) females, but without dose dependency and similar results for the hindlimbs or during the modified Irwin test, this variety was not considered to be a test item related effect.
The total travelled distance in the High dose group and the shape of the curve of activity in 5 minute periods over one hour, were comparable to the control groups for both sexes; all data was within the normal expected range. Sporadic statistical significant differences were of no toxicological relevance. There was no test item related effect on locomotor activity in either sex.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
- Found dead animals:
Treatment-related slight centrilobular hepatocellular hypertrophy of the liver was observed in 1 out of 4 female. Various microscopic findings induced by stress due to treatment such as slight decrease of white pulp size/cellularity in the spleen and moderate decrease size/cellularity of the thymic cortex (correlated with gross changes), slight decrease of myeloid cellularity in the femoral and sternal bone marrows, slight diffuse vacuolation of the adrenal’s medulla were present in this found dead female. In addition, moderate decrease of white pulp size/cellularity in the spleen was also recorded in one other female rat. There were no findings that clearly indicated a cause of death.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive ability assessment and indices:
There were no differences between the control and test substance treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the test substance administration. The mating indices were normal in all groups (93-100%). The fertility index was 83, 100, 83 and 92% for males, and 83, 100, 83, 100% in females in the Control, Low, Mid and High dose groups, respectively. The gestation index was 100% in all groups.

Test substance administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 4 days of pairing (cohabitation). In one High dose female (ID 4507), the duration of the mating period was 14+4 days, with continuous dioestrus observed during the oestrus cycle, however after the new male was introduced after Day 14, this female successfully paired on Day 18 of the mating period (after 4 days). Therefore, the elongated mating period may have been partly due to the male animal (ID 4007), where this variation was regarded as individual, normal biological variability and without toxicological significance.

Mean pre-coital interval was 2.2, 2.5, 2.8 and 2.4 days in Control, Low, Mid and High dose groups, respectively.

Evaluation of the gestation, parturition and post-partal period:
There was no effect of treatment noted during gestation, parturition or the post-partal period. Delivery lasted more than three hours in one Low (100 mg/kg bw/day) and one High (1000 mg/kg bw/day) dose animal. These relatively long parturition durations were considered to be incidental, and not related to the treatment. Statistically shorter duration of pregnancy occured in the Low (100 mg/kg bw/day, p<0.01) and High (1000 mg/kg bw/day, p<0.05) groups, but all animals in the study had a pregnancy duration of 22 or 23 days, so all were considered normal. The statistical difference was considered incidental and unrelated to the treatment. The number of corpora lutea and number of implantation sites was comparable to the control mean at all dose groups. There were no significant differences or effects that could be ascribed to treatment on the pre-implantation, intrauterine, post-natal or total mortality values (litter mean and %) at up to and including 1000 mg/kg bw/day.

For details refer to 'any other information on results incl. tables'

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: for systemic effects

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were comparable to control values at up to and including 1000 mg/kg bw/day. Overall, there were no treatment-related effects on pup mortality. There were no treatment related effect on the viability of pups on PND0 and PND 4.

Description (incidence and severity):

There were no effects considered adverse on the offspring weight or weight gain following administration of the test substance at 100, 300 or 1000 mg/kg bw/day to parental generation under the conditions of this study. When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND 0 and 4 showed no statistically or toxicologically significant differences compared to controls in the F1 generation.

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic findings were recorded.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

 Summary of reproductive parameters, females

Parameters	Groups/Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of treated animals	12	12	12	15
Number of preterminal death (found dead and preterminal euthanasia)	0	0	0	5
Number of mated animals	12	12	12	12*
Number of sperm positive females	12	12	12	12**
Number of females with corpora lutea,  but no implantation sites	2	0	2	0
Number of pregnant females	10	12	10	10
Number of pregnant females with live born(s)	10	12	10	10
Female mating index (%)	100	100	100	100
Female fertility index (%)	83	100	83	100
Female gestation index (%)	100	100	100	100

*One additional paired female animal (No. 4503) was found dead during the mating period, and therefore was excluded from further analysis.

** For two sperm positive females, mortality occurred during the early stages of pregnancy, therefore fertility index was calculated after these animals were excluded from sperm positive females (number of animals included for further calculations: 10).

 

Summary of the intrauterine evaluation

Parameters	Groups/Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of evaluated females	12	12	12	10
Mean number of corpora lutea	13.00	15.83	14.25	14.60	NS
Pre-implantation mortality, mean	1.67	0.00	1.50	0.20	NS
Pre-implantation mortality (%), mean	18.33	0.00	16.67	1.05	NS
Mean number of implantations	11.33	15.83	12.75	14.40	NS
Number of evaluated litters	10	12	10	10
Intrauterine mortality, mean	0.40	0.67	0.50	0.50	NS
Intrauterine mortality (%), mean	4.81	4.05	3.20	5.64	NS
Post-natal mortality, mean	0.10	0.25	0.20	0.20	NS
Post-natal mortality (%), mean	0.71	1.47	1.21	3.33	NS
Total mortality, mean	0.50	0.92	0.70	0.70	NS
Total mortality (%), mean	5.52	5.44	4.35	7.84	NS
Duration of pregnancy (days)	22.50	22.00**	22.30	22.10*	DN
Number of pups born, mean	13.20	15.42	14.90	14.00	NS
Number of live born, mean	13.20	15.17	14.80	13.90	NS

Notes:

Mean values were rounded to two decimal places.

* = p<0.05, ** = p<0.01

DN = Duncan's Multiple Range Test

NS = Non Significant

 

Summary of reproductive parameters, males

Parameters	Groups/Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of treated animals	12	12	12	15
Number of preterminal death (found dead)	0	0	0	1
Number of mated animals	12	12	12	13
Number of infertile animals	0	0	0	1
Male mating index (%)	100	100	100	93
Male fertility index (%)	83	100	83	100

Note:

Male fertility figures takes account only of males where the mated female survived to the end of gestation.

Conclusions:

Under the study conditions and in absence of adverse effects, the rat NOAEL for reproductive and development effects can be considered to be at 1000 mg/kg bw/day.

Executive summary:

A screening study was conducted to determine the reproductive and development toxicity of the test substance according to OECD guideline 422. The general systemic toxic potential of the substance to Wistar rats was assessed by daily oral administration (gavage). Three groups of 12 male and 12 female rats received the test substance at doses of 0, 100, 300 or 1000 mg/kg bw/day. The males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating), and females for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 days post-partum dosing). A similarly constituted control group received the vehicle, propylene glycol, at the same volume-dose. During the study, apart from systemic parameter examinations data was recorded reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals. Organ weight (including gonads), macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.Daily administration of the test substance at dose levels of 100 or 300 mg/kg bw/day did not result in adverse changes in clinical signs, neurological assessment, body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters. Test substance related mortality (approximately 15% mortality) was observed in the High dose group (1000 mg/kg/d) and clinical adverse effects were present in High dose females. There were no effects on surviving animals on body weight or clinical pathology. Test substance related effects in liver and kidney in the High dose group were considered to be an adaptive change and not an adverse effect of treatment. Further, no test substance-related microscopic changes were noted in the reproductive organs in any groups.

Under the study conditions, the rat NOAEL for reproductive and development effects was established at 1000 mg/kg bw/day and at 300 mg/kg bw/day for systemic effects (Hargitai, 2016).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 26, 2016 to March 24, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

but considered not to adversely affect the results or integrity of the study.

Qualifier:

according to guideline

Guideline:

other: OECD guidance document 43

Version / remarks:

The guideline was designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.

Principles of method if other than guideline:

- To obtain information on the possible toxic effects of the test substance following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels. A control group received the vehicle only (propylene glycol).
- The study included a reproductive/ developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance including gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 4 post-partum.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on test animals or test system and environmental conditions:

The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany)
Housing conditions: SPF at the supplier, standard laboratory conditions during the study
Number of animals: 51 male, 51 female rats, 4 groups. 12 animals/sex/group, with the exception of the High dose group, where 15 animals/sex/group was used. Animals originated from different units, to avoid brother/sister matings
Age of animals: young adult rats, at least 10 weeks old at starting and 12 weeks at mating
Body weight at the start: males: 362-422 g, females: 218-261 g
Acclimation period: at least 5 days
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.2-24.3 °C (target: 22 ± 3°C)
Relative humidity: 40 - 62% (target: 30 - 70%)
Ventilation: 15-20 air exchanges/hour
Food and water supply: ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" and tap water, ad libitum

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

Analysis of test substance formulations for concentration and homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken and analysed from test substance formulations and all concentrations. Sample analysis was performed on 3 occasions (with an additional occasion for the High dose (250 mg/ml) formulation) for homogeneity (top, mid and bottom) and all prepared formulations were analysed for concentration. One set was collected for analysis and one set as a back-up. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of test substance. Description of the analytical method and the results of the formulation analysis are included in the Analytical report provided by the Analytical Laboratory of CiToxLAB Hungary Ltd.
Analysis of the test substance formulation samples of 1-250 mg/mL concentration range showed no decrease of concentration and can be considered as stable for 7 days at room temperature.
The measured concentrations of the test substance evaluated for each test substance-dose group varied between 94 % and 107 % of the nominal contents. No test substance was detected in the control samples. These results were within the acceptable range (85% - 115%) and are acceptable for the study purposes. All samples were found to be homogeneous.

Details on mating procedure:

Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male from the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 14 days. If the first 14 days of the mating was unsuccessful, then the female was paired for a second period with a proven male from the same dose group (1:1 mating).
A vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.

Duration of treatment / exposure:

- Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating), and then euthanized and subjected to necropsy examination.
- Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. (Females showing no-evidence of copulation were sacrificed 25 days after the end of the mating period (two animals in the Control and two animals in the Mid dose, with no delivery).
- All F1 offspring were terminated on Day 4 post-partum. In order to allow for overnight fasting of dams prior to urine collection on PPD5, the offspring were euthanized on PPD/PND 4 and the dams on PPD/PND 5.

Frequency of treatment:

daily on a 7 days/week basis by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 4 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on most recent individual body weights. (Dosing of both sexes began after at least 5 days of acclimation and 2 weeks before mating and continued up to the day before necropsy).

Duration of test:

- Age at mating of the mated animals in the study: F0 generation: Males: ca. 12 weeks ; Females: ca. 12 to 14 weeks. (i.e. after two weeks treatment).

Dose / conc.:

0 mg/kg bw/day

Remarks:

Concentration: 0 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

100 mg/kg bw/day

Remarks:

Concentration: 25 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

300 mg/kg bw/day

Remarks:

Concentration: 75 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Concentration: 250 mg/mL
Dose volume: 4 mL/kg bw

No. of animals per sex per dose:

at least 12

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work including the results of a repeated dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 15/340-220PE). The aim was to induce toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
Based on the results from these preliminary studies, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study.

Maternal examinations:

Refer to the study under repeated dose endpoint (section 7.5.1) for details on systemic parameter examinations.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes. At termination detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]

Statistics:

Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, or using the software PROVANTIS v.9, as appropriate. Group means and standard deviations were calculated from numerical data obtained in the study.
The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) by an appropriate statistical method (Bartlett, ANOVA/ANCOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests, T-test, Wilcoxon test, Chi2 test). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA/ANCOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test or Kruskal-Wallis test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible. For SMART evaluation, T-test was used

Indices:

Observations: mean pup body weight (per pup within the group and per litter) on PND0 and PND4, mean pup body weight gain (per litter) between postnatal Days 0-4, number of live births per litter, and number of viable pups per litter on PND0 and PND4. Survival Index of pups on postnatal Days 0 and 4 and sex ratio % (on postnatal Days 0 and 4)
Offspring viability indices including; (post-implantation) survival index (and/or viability index and live birth index can be inferred), pre-implantation mortality, intrauterine mortality, total mortality and sex ratio.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
Terminally euthanised animals:
- Male animals: no clinical signs were detected in the Control, Low and Mid dose groups during the study. In the High (1000 mg/kg bw/day) dose group liquid faeces, noisy respiration and piloerection were occasionally present up to three animals during the treatment. These observations were considered to be treatment related.
- Female animals: thin fur/alopecia was observed in all groups, peaking at the end of the experiment, but is not considered to be related to test substance. No clearly adverse effects of the test substance related were noted in the Low dose groups during the study. In the Mid (300 mg/kg bw/day) dose group, piloerection and noisy respiration was observed sporadically in just 1 or 2 females. These non-specific observations seen sporadically at a low incidence is not considered to be a clear sign of an adverse effect of the test substance. In the High (1000 mg/kg bw/day) dose group, the non-specific signs of piloerection and noisy respiration was seen in a small number of male and the majority of females. Hunched back was also present in up to three female animals, which may be treatment related. A 1 to 2 cm wide crust was seen on the skin for five days in one animal, yellow coloured discharge in one animal, and discharge from vagina in one animal was observed during the treatment; these findings are of uncertain relationship with treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
A test substance related mortality (approximately 15% mortality) was observed in the High dose group (1000 mg/kg bw/day). One male (Day 16) and 4 females (Days 3, 6, 18 and 22 ) of the High dose group were found dead. In addition, one female (Day 17) was preterminally euthanized.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance related effects were noted on the mean body weight and body weight gain values following daily administration of the test substance at dose levels up to and including 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance related differences in the mean daily food consumption in any test substance treated group when compared to the controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
When compared to the controls, there were no differences that could be considered toxicologically significant in the Low and Mid dose (100 and 300 mg/kg bw/day, respectively) groups. The High (1000 mg/kg bw/day) males only had a slightly but significantly (p<0.05) lower Mean Cell Haemoglobin Concentratin (MCHC). The female High dose group had a slightly but significantly (p<0.05) longer Activated Partial Thromboplastin Time (APTT). However the data from all animals was in the normal range and these small differences were not consistent between sexes and were not considered to be an adverse effect of treatment.
For other parameters, evaluation of the mean and individual results in comparison with the control data did not reveal any test-substance related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range and of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
In the animals evaluated at termination (on Day 28 in males and on PPD5 in females), there were no clearly toxicologically significant changes or adverse effects on the animal serum chemistry that could be ascribed to the test substance administration in the conditions of this study. Urea and chloride concentrations were statistically significantly higher in the male High (1000 mg/kg bw/day) dose group compared to the Control (p<0.05 and p<0.01, respectively). The female High dose group had statistically significantly (p<0.05) lower phosphorus values. However the data for these 3 parameters from all animals was in the normal range and these small differences were not consistent between sexes and were not considered to be an adverse effect of treatment.
Some differences in the Low and Mid treated groups also attained statistical significance; however, there was no dose or gender response or the values were within the physiological ranges. For this reason, these variations were not considered toxicologically significant or related to treatment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
A dose dependent decrease of pH was observed in both sexes, reaching statistical significant difference compared to the control value at the High (1000 mg/kg bw/day) dose males (p<0.05) and females (p<0.01). In the High dose only, slight but statistical significance differences were recorded with lower pH and higher urine gravity in both sexes (p<0.01) and lower urine volume in makes only (p<0.05). However, all values were in the normal control range, these findings were regarded as minor variations and were not considered to indicate an adverse effect of the test substance.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
Kidney weights were slightly higher than control in both sexes at the High dose (by 8% in males and by approximately 10% in females). This was statistically significant both in terms of absolute weight or when adjusted for body or brain weights. There were also slight differences in urine analysis at the High dose level in both sexes, so the small organ weight differences may reflect an adaptive or functional difference. A similar statistically significant difference was seen in female kidney weights in the Mid dose group but the data was all in the normal range. Therefore it was not considered to be a clear effect of the test substance. At the Mid and Low dose levels, it is considered that minor statistical differences did not reflect an effect of the test susbtance. In the absence of any histopathological or blood chemistry changes, there is no evidence of an adverse effect in the kidneys at any dose level.
In females, increased liver weights were seen in the High dose group only (by 12% absolute weight). Increased liver weights were not seen in the males of the High dose group. These liver weight changes correlate well with the centrilobular hepatocellular hypertrophy observed in High (1000 mg/kg bw/day) dose female animals.
There were no other toxicologically significant differences among groups in the weights of other organs measured when compared to controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
- Found dead animals: Small spleen in 2 out of 4 found dead females and small thymus in 1 out of 4 female corresponded with findings noted during microscopic examination.
- Preterminal euthanasia: Small spleen and thymus, pale foci of the left lobe in the liver were observed at necropsy and were related to the treatment. Dilatation (with gas) of the oesophagus, stomach, duodenum, jejunum, ileum, caecum and colon, and red/clear material at the perinasal fur did not have relationship to the treatment.
- Terminal animals: No treatment-related macroscopic findings were observed up to the dose level of 1000 mg/kg bw/day.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
Neurological:
There were no toxicologically significant changes in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test groups.
There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. Grip strengths of the forelimb were significantly (p <0.05) lower in Mid dose (300 mg/kg bw/day) females, but without dose dependency and similar results for the hindlimbs or during the modified Irwin test, this variety was not considered to be a test item related effect.
The total travelled distance in the High dose group and the shape of the curve of activity in 5 minute periods over one hour, were comparable to the control groups for both sexes; all data was within the normal expected range. Sporadic statistical significant differences were of no toxicological relevance. There was no test item related effect on locomotor activity in either sex.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results
- Found dead animals:
Treatment-related slight centrilobular hepatocellular hypertrophy of the liver was observed in 1 out of 4 female. Various microscopic findings induced by stress due to treatment such as slight decrease of white pulp size/cellularity in the spleen and moderate decrease size/cellularity of the thymic cortex (correlated with gross changes), slight decrease of myeloid cellularity in the femoral and sternal bone marrows, slight diffuse vacuolation of the adrenal’s medulla were present in this found dead female. In addition, moderate decrease of white pulp size/cellularity in the spleen was also recorded in one other female rat. There were no findings that clearly indicated a cause of death.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no reported effects on: reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the test substance administration. There were additionally no reported affects on male or female reproductive organs or for example: sperm measures.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no significant differences or effects that could be ascribed to treatment on the pre-implantation, intrauterine, post-natal or total mortality values (litter mean and %) at up to and including 1000 mg/kg bw/day.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

It was considered offspring survival was not affected by treatment.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Litter size, offspring survival was not affected by treatment.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation length (and/or gestation index) was considered unaffected by treatment.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

It was considered estrous cyclicity, pre-coital interval, fertility and mating performance were unaffected by treatment. Litter size, offspring survival was not affected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: for systemic effects

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: for reproductive performance and/or developmental effects

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring survival

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring sex ratio

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring litter size or offspring body weight

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring survival

External malformations:

no effects observed

Description (incidence and severity):

Ano-genital distances for male or female offspring were unaffected by treatment with test item.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring did not reveal any findings attributable to treatment.

Visceral malformations:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring did not reveal any findings attributable to treatment.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Summary of reproductive parameters, females

Parameters	Groups/Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of treated animals	12	12	12	15
Number of preterminal death (found dead and preterminal euthanasia)	0	0	0	5
Number of mated animals	12	12	12	12*
Number of sperm positive females	12	12	12	12**
Number of females with corpora lutea,  but no implantation sites	2	0	2	0
Number of pregnant females	10	12	10	10
Number of pregnant females with live born(s)	10	12	10	10
Female mating index (%)	100	100	100	100
Female fertility index (%)	83	100	83	100
Female gestation index (%)	100	100	100	100

*One additional paired female animal (No. 4503) was found dead during the mating period, and therefore was excluded from further analysis.

** For two sperm positive females, mortality occurred during the early stages of pregnancy, therefore fertility index was calculated after these animals were excluded from sperm positive females (number of animals included for further calculations: 10).

 

Summary of the intrauterine evaluation

Parameters	Groups/Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of evaluated females	12	12	12	10
Mean number of corpora lutea	13.00	15.83	14.25	14.60	NS
Pre-implantation mortality, mean	1.67	0.00	1.50	0.20	NS
Pre-implantation mortality (%), mean	18.33	0.00	16.67	1.05	NS
Mean number of implantations	11.33	15.83	12.75	14.40	NS
Number of evaluated litters	10	12	10	10
Intrauterine mortality, mean	0.40	0.67	0.50	0.50	NS
Intrauterine mortality (%), mean	4.81	4.05	3.20	5.64	NS
Post-natal mortality, mean	0.10	0.25	0.20	0.20	NS
Post-natal mortality (%), mean	0.71	1.47	1.21	3.33	NS
Total mortality, mean	0.50	0.92	0.70	0.70	NS
Total mortality (%), mean	5.52	5.44	4.35	7.84	NS
Duration of pregnancy (days)	22.50	22.00**	22.30	22.10*	DN
Number of pups born, mean	13.20	15.42	14.90	14.00	NS
Number of live born, mean	13.20	15.17	14.80	13.90	NS

Notes:

Mean values were rounded to two decimal places.

* = p<0.05, ** = p<0.01

DN = Duncan's Multiple Range Test

NS = Non Significant

 

Summary of reproductive parameters, males

Parameters	Groups/Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of treated animals	12	12	12	15
Number of preterminal death (found dead)	0	0	0	1
Number of mated animals	12	12	12	13
Number of infertile animals	0	0	0	1
Male mating index (%)	100	100	100	93
Male fertility index (%)	83	100	83	100

Note:

Male fertility figures takes account only of males where the mated female survived to the end of gestation.

Conclusions:

Under the study conditions and in absence of adverse effects, the rat NOAEL for reproductive and development effects can be considered to be at 1000 mg/kg bw/day.

Executive summary:

A screening study was conducted to determine the reproductive and development toxicity of the test substance according to OECD guideline 422. The general systemic toxic potential of the substance to Wistar rats was assessed by daily oral administration (gavage). Three groups of 12 male and 12 female rats received the test substance at doses of 0, 100, 300 or 1000 mg/kg bw/day. The males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating), and females for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 days post-partum dosing). A similarly constituted control group received the vehicle, propylene glycol, at the same volume-dose. During the study, apart from systemic parameter examinations data was recorded reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals. Organ weight (including gonads), macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.Daily administration of the test substance at dose levels of 100 or 300 mg/kg bw/day did not result in adverse changes in clinical signs, neurological assessment, body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters. Test substance related mortality (approximately 15% mortality) was observed in the High dose group (1000 mg/kg/d) and clinical adverse effects were present in High dose females. There were no effects on surviving animals on body weight or clinical pathology. Test substance related effects in liver and kidney in the High dose group were considered to be an adaptive change and not an adverse effect of treatment. Further, no test substance-related microscopic changes were noted in the reproductive organs in any groups.

Under the study conditions, the rat NOAEL for reproductive and development effects was established at 1000 mg/kg bw/day and at 300 mg/kg bw/day for systemic effects (Hargitai, 2016).

Cross-reference 3

Reason / purpose for cross-reference:

other:

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

From November 26, 2015 to December 10, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Principles of method if other than guideline:

This non-GLP preliminary dose range finding study did not follow a specific OECD guideline, but it was designed to allow selection of appropriate dose levels for the upcoming OECD No. 422 study. This study was conducted in accordance with a written study plan; it was authorized by the Sponsor and test facility management, and followed the applicable Standard Operating Procedures.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:Wi rats

Details on species / strain selection:

The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany)
Housing conditions: SPF at the supplier, standard laboratory conditions during the study
Number of animals: 20 male and 20 female rats were used in the study. The spare animals (4 animals/sex) were assigned to the spare colony after the completion of the study
Age of animals: young adult rats, 11 weeks old at the start
Body weight at the start: males: 381 - 431 g; females: 243 -287 g
Acclimation period: 7 days
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.2-24.3 °C (target: 22 ± 3°C)
Relative humidity: 40 - 62% (target: 30 - 70%)
Ventilation: 15-20 air exchanges/hour
Food and water supply: ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" and tap water, ad libitum

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

Five males and five females per group were treated in each group daily for 14 consecutive days in order to obtain preliminary information on the potential toxicity of the test substance following repeated administration at dose levels of 100, 300 and 1000 mg/kg bw/day. The control group was treated concurrently with the vehicle only (propylene glycol). The first day of dosing of each animal was regarded as Day 0. A constant dose volume of 4 mL/kg body weight was administered to all animals. The individual volume of the treatment was based on the most recent individual body weight of the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analysis of test substance formulations for concentration and/or homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. using a validated HPLC method (CiToxLAB study code 15/340-316AN). Top, middle and bottom duplicate samples were taken from test substance formulations once during the study, one set to analyze and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken in duplicate from the middle of the vehicle control solution for concentration measurement.
- Concentration and homogeneity of the dosing formulations were determined once during treatment period. No test substances were detected in the negative (vehicle) control sample. All test substance formulations were shown to be homogeneous and they were found to be in the range of 91 to 101% of nominal concentrations. Based on these results, formulations were considered suitable for the study purposes.

Duration of treatment / exposure:

14 consecutive days

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Concentration level: 0 mg/mL
Dose Volume: 4 mL/kg bw

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Concentration level: 025 mg/mL
Dose Volume: 4 mL/kg bw

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Concentration level: 75 mg/mL
Dose Volume: 4 mL/kg bw

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Concentration level: 250 mg/mL
Dose Volume: 4 mL/kg bw

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose levels: The dose levels were set by the Sponsor based on available data and information from previous experimental work, including the results of an acute oral toxicity study and of a Maximum Tolerated Dose (MTD) finding study with this test substance.

- Animal assignment to the study and randomisation: All animals were weighed on the day before the start of the treatment period and randomly allocated to study groups. The results of the randomization were checked using a computer program to verify the homogeneity and deviations between the groups. Males and females were randomised separately.

Positive control:

-

Observations and examinations performed and frequency:

Mortality and clinical observations:
- Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
- Clinical observations were made twice daily, before and after treatment, at the beginning and towards the end of the working day as practical. The animals were monitored for any clinical signs, including pertinent behavioural changes, signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep or coma.

Body weight and food consumption:
- Body weight of each animal was recorded with precision of 1 g at randomization (Day 0), then on Days 3, 7, 10, 13 and 14 (fasted, prior to necropsy). In case of High dose males, Day 1 values were also recorded (these values are kept in the raw data binder and will be archived, but not reported).

Food consumption:
- It was recorded with precision of 1 g at the start (Day 0) and then on Days 3, 7, 10 and 13.

Haematology: See under 'Any other information on materials incl. tables'

Clinical chemistry: See under 'Any other information on materials incl. tables'

Sacrifice and pathology:

Clinical pathology:
- On Day 14, after an overnight period of food deprivation of animals, blood samples were collected from all animals immediately prior to the scheduled necropsy by heart puncture under pentobarbital anaesthesia. Two blood samples were collected for clinical pathology evaluation: one for haematology (in tubes with K3-EDTA, 1.6 mg/mL blood) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.
Pathology:
- For termination on Day 14, animals were euthanized under pentobarbital anaesthesia by exsanguination. After exsanguination, the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
- Organs were trimmed of fat and weighed in all animals after completion of the 14-day treatment. Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported. For the list of organs which were measure see under 'any other information on materials and methods incl. tables'
- Histopathology: on completion of the macroscopic examination, tissues and organs were retained from all animals. (The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution; all other organs were retained in 10% buffered formalin solution.) For details on tissue and organs retained from animals see under 'any other information on materials and methods incl. tables'

Statistics:

- Data was collected using the PROVANTIS software or recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the PROVANTIS software, Microsoft Office Word and/or Excel, as appropriate.
- Statistical evaluation of data as applicable was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest).
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not show a normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the High dose group (1000 mg/kg bw/day) the following observations were recorded during the observation period: hunched back in one male and in three females, piloerection in one female, distended abdomen in two females, slightly to moderately decreased activity in five males and in three females. These observations were considered as being test substance related
Other observations, such as slightly noisy respiration and/or dyspnoea in one male and in three females, and prolapsed uterus in one female, were not considered to be systemic effects of the test substance. There were no relevant clinical observations in the Low or Mid dose groups.

Mortality:

no mortality observed

Description (incidence):

There was no unscheduled mortality during the study

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- There was no statistically significant effect on the body weights of the test substance treated groups (males/females) when compared to the control at any occasion, except in the High dose group females, where significantly (p<0.05) lower mean body weight (by approx. 14%) was recorded at the end of the treatment period (Day 13).
- The calculated body weight gain values for the entire treatment period of the study (Day 0-13) were significantly lower for High dose males and females (p<0.01 and p<0.05, respectively) when compared to the control animals, although in case of the High dose males, the effect was largely due to an extreme value of one animal. The significant difference (p<0.05) was also observed in the periods of Day 0-3 and Day 7-10 in High dose males. These findings on the body weight and body weight gain values of the High dose animals were considered as treatment related changes. Besides these observations, significantly lower body weight gain (p<0.05) was recorded for Mid dose males in the period of D7-10 when compared to the control animals, however this temporary difference had no impact on the total gain during the entire period of the study, so it was considered as not related to the treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In High dose (1000 mg/kg bw/day) males and females, decreases were observed in the mean daily food consumption in some periods when compared to the control animals, although statistical analysis of the data could not be performed (animals were group caged).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase (p<0.05) was observed in the mean platelet volume (p<0.05) of High dose males (1000 mg/kg bw/day). However, as the observed value was within the historical control range, this fact was considered to have no toxicological significance.
One female in the High dose group (1000 mg/kg bw/day) showed unusual haematology values in the cellular ratio and this caused significant differences between the High dose group and the Control group in some parameters: decreased (p<0.05) relative amount of lymphocytes and increased (p<0.01) relative amount of monocytes. However, as values of the other four High dose females were within the historical control range, no clear relationship to the treatment can be confirmed in this case.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Male animals: increased (p<0.05) creatinine concentration in the High dose group (1000 mg/kg bw/day); increased total protein and albumin concentrations (p<0.05) in the Mid dose group (300 mg/kg bw/day), decreased alkaline phosphatase activity in the Mid dose group (p<0.05) and in the High dose group (p<0.01). However, all these findings were without consistent patterns and/or without dose response; and correlated with the existing historical control database. Thus, they were considered as incidental or individual findings with no toxicological significance.
- Female animals: increased urea concentration (p<0.01) in the High dose group (1000 mg/kg bw/day). The observed value was slightly higher than the upper limit of the historical control range. Furthermore, increased value was also recorded in the Mid dose group (300 mg/kg bw/day), although it did not gain statistical significance. However, other parameters of kidney function were not affected, so this fact was considered to have no clear toxicological relevance.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- Male animals: significantly (p<0.05) increased liver absolute weights in the Low and Mid dose groups (100 and 300 mg/kg bw/day, respectively), and significantly (p<0.05) lower absolute weight of epididymides in the High dose group (1000 mg/kg bw/day) when compared to the control absolute weight values. Nevertheless, the changes were non-significant when the relative (to body or brain weight) were compared. Since there was a body weight difference between groups, the relative weights are considered to be a more reliable indicator of any treatment relationship.
- Female animals: dose dependent decrease of spleen and thymus absolute and relative weights, which reached significant levels in the High dose group animals (p<0.01 and p<0.05, respectively). However, the absolute values were within the historical control range in all case except of the absolute spleen weight. Two out of five animals in the High dose group has spleen weights which were below the other groups including control. One female had a particularly low spleen weight, associated with an unusual haematology profile; findings in this female were not attributed to treatment. Furthermore, the statistical differences at the High dose group were generally related to the body weight decrease.
In conclusion, based on these observations (lack of dose response and/or lack of similar trends in the other sex, relevance with the existing historical control data), these findings were considered not attributed to the test substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- In the High dose males (1000 mg/kg bw/day) the following observations were recorded at necropsy: in the stomach, diffuse thickness of the glandular mucosa, many, depressed, dark red areas on the glandular mucosa, raised area on the non-glandular mucosa or gastric dilatation. In the High dose females (1000 mg/kg bw/day), gastric dilatation was recorded in two animals, diffuse thickness of the glandular mucosa and many, depressed, dark red areas on the glandular mucosa was also observed. These gastrointestinal observations recorded in the High dose males and females were considered to be test substance related. Other changes (small spleen in one animal, small thymus was recorded in two animals were considered to be incidental.
- In the Mid dose females (300 mg/kg bw/day), gelatinous material was observed in the vagina, and the left uterine horn was missing in one animal. These observations were considered as not attributed to the test substance.
- No findings were recorded in Low dose (100 mg/kg bw/day) animals.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

no

Conclusions:

Under the study conditions, the NOEL for systemic toxicity in rats following 14 days of treatment was established at 300 mg/kg bw/day.

Executive summary:

A dose range finding study was conducted to obtain preliminary information about the toxic potential of the test substance in Wistar rats. In this study, three groups each comprising five male and five female rats received the test substance at doses of 0, 100, 300 or 1000 mg/kg bw/day. Control animals received the vehicle (propylene glycol) alone. Animals were treated daily for 14 consecutive days. Mortality checking and clinical observations were performed twice daily. Body weight and food consumption were measured for all animals on Days 0, 3, 7, 10, 13 and prior to scheduled necropsy on Day 14. Following the daily repeated dose administration for 14 days, blood samples were collected for clinical pathology at necropsy. Gross macroscopic examination was performed at necropsy at the termination, one day after the last treatment. Selected organs were weighed, and selected tissues were preserved by fixation.

No unscheduled mortality occurred during the study. The test substance caused non-specific clinical signs such as decreased motility in the High dose group (1000 mg/kg bw/day). No test substance related systemic effects were seen in the Low and Mid dose groups (100 and 300 mg/kg bw/day, respectively). A drop in body weight was recorded in the High dose (1000 mg/kg bw/day) males and females towards the end of the treatment period. Similarly, significant effect on the body weight gain was observed for High dose males and females. The food intake values reflected the body weight differences. These findings on the body weight, body weight gain and food intake values of the High dose animals were considered as treatment-related changes. There were no clear toxicologically significant effects at any dose levels in the haematology or clinical chemistry parameters examined on Day 14. Test substance-related changes were observed in the stomachs of some rats of each sex at the High dose (l000 mg/kg bw/day). Thickened/raised areas of the non-glandular stomach mucosa, multifocal depressed red areas of the non-glandular stomach mucosa, and dilated stomach/caecum/colon were considered to be treatment related effects. No relevant findings were recorded in Mid or Low dose animals (100 and 300 mg/kg bw/day). No toxicologically relevant differences were observed in the organ weight of any dose groups.

Under the study conditions, the NOAEL for systemic toxicity in rats following 14 days of treatment was 300 mg/kg bw/day (Hargitai, 2016).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

but considered not to adversely affect the results or integrity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD guidance document 43

Version / remarks:

The guideline was designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.

Principles of method if other than guideline:

- To obtain information on the possible toxic effects of the test substance following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels. A control group received the vehicle only (propylene glycol).
- The study further included a reproductive/ developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance including gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 4 post-partum.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(phosphonooxy)ethyl methacrylate

EC Number:

246-342-6

EC Name:

2-(phosphonooxy)ethyl methacrylate

Cas Number:

24599-21-1

Molecular formula:

C6H11O6P

IUPAC Name:

{2-[(2-methylprop-2-enoyl)oxy]ethoxy}phosphonic acid

Constituent 2

Reference substance name:

Bis(methacryloyloxyethyl) hydrogen phosphate

EC Number:

251-040-2

EC Name:

Bis(methacryloyloxyethyl) hydrogen phosphate

Cas Number:

32435-46-4

Molecular formula:

C12H19O8P

IUPAC Name:

bis({1-[(2-methylprop-2-enoyl)oxy]ethoxy})phosphinic acid

Constituent 3

Reference substance name:

2-[bis[2-(2-methylprop-2-enoyloxy)ethoxy]phosphoryloxy]ethyl 2-methylprop-2-enoate

Molecular formula:

C18H27O10P

IUPAC Name:

2-[bis[2-(2-methylprop-2-enoyloxy)ethoxy]phosphoryloxy]ethyl 2-methylprop-2-enoate

Constituent 4

Reference substance name:

Pyrophosphate mixture

Molecular formula:

UVCB substance, molecular formula for applicable

IUPAC Name:

Pyrophosphate mixture

Constituent 5

Reference substance name:

Orthophosphoric acid

EC Number:

231-633-2

EC Name:

Orthophosphoric acid

Cas Number:

7664-38-2

Molecular formula:

H3O4P

IUPAC Name:

phosphoric acid

Test material form:

liquid: viscous

Details on test material:

PARAD substance 139

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany)
Housing conditions: SPF at the supplier, standard laboratory conditions during the study
Number of animals: 51 male, 51 female rats, 4 groups. 12 animals/sex/group, with the exception of the High dose group, where 15 animals/sex/group was used. Animals originated from different units, to avoid brother/sister matings
Age of animals: young adult rats, at least 10 weeks old at starting and 12 weeks at mating
Body weight at the start: males: 362-422 g, females: 218-261 g
Acclimation period: at least 5 days
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.2-24.3 °C (target: 22 ± 3°C)
Relative humidity: 40 - 62% (target: 30 - 70%)
Ventilation: 15-20 air exchanges/hour
Food and water supply: ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" and tap water, ad libitum

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

The oral route was selected, as it is a possible route of exposure to the test substance in humans.

The test substance was formulated in the vehicle, as a clear solution at the appropriate concentrations according to the selected dose level and volume, in the Pharmacy of CiToxLAB Hungary Ltd.

Formulations were prepared in up to 6-day intervals and stored at room temperature, based on the stability assessment results. Stability of the test substance in the vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to CiToxLAB study code 15/340-316AN). Analysis of PARAD substance 139 formulation samples of 1-250 mg/mL concentration range showed no decrease of concentration and can be considered as stable for 7 days at room temperature.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

Analysis of test substance formulations for concentration and homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken and analysed from test substance formulations and all concentrations. Sample analysis was performed on 3 occasions (with an additional occasion for the High dose (250 mg/ml) formulation) for homogeneity (top, mid and bottom) and all prepared formulations were analysed for concentration. One set was collected for analysis and one set as a back-up. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of test substance. Description of the analytical method and the results of the formulation analysis are included in the Analytical report provided by the Analytical Laboratory of CiToxLAB Hungary Ltd.
Analysis of the test substance formulation samples of 1-250 mg/mL concentration range showed no decrease of concentration and can be considered as stable for 7 days at room temperature.
The measured concentrations of the test substance evaluated for each test substance-dose group varied between 94 % and 107 % of the nominal contents. No test substance was detected in the control samples. These results were within the acceptable range (85% - 115%) and are acceptable for the study purposes. All samples were found to be homogeneous.

Duration of treatment / exposure:

- Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating), and then euthanized and subjected to necropsy examination.
- Females were dosed for 54 days (14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.

Frequency of treatment:

Daily on a 7 days/week basis by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 4 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on most recent individual body weights. (Dosing of both sexes began after at least 5 days of acclimation and 2 weeks before mating and continued up to the day before necropsy.)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Concentration: 0 mg/mL
Dose volume: 4 mL/kg bw

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Concentration: 25 mg/mL
Dose volume: 4 mL/kg bw

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Concentration: 75 mg/mL
Dose volume: 4 mL/kg bw

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Concentration: 250 mg/mL
Dose volume: 4 mL/kg bw

No. of animals per sex per dose:

at least 12

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work including the results of a repeated dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 15/340-220PE). The aim was to induce toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
Based on the results from these preliminary studies, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study.

Positive control:

-

Examinations

Observations and examinations performed and frequency:

Clinical signs and Functional observation battery:
- Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, and/or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment. All animals were also monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable.
- Detailed examinations were performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were performed outside the home cage in a standard arena, at similar day times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

- Neurotoxicity (5 males and 5 females per group):
Assessment of potential test substance related neurotoxicity was performed in the morning and prior to dosing, during the last exposure week (males on Day 24; females on PPD 4). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength (manual and instrumental) and measurement of landing foot splay and fore/hind limb grip strength. Qualitative and quantitative assessments of motor activity were performed. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated using a scoring system, where score 0 was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an untreated animal.

- Body weight measurement:
All adult animals were weighed with an accuracy of 1 g for randomization purposes, then on Day 0, at least weekly thereafter and at termination. Parent females were weighed on gestation Days GD0, 3, 7, 14 and 20 and on post-partum Days PPD0 (within 24 hours after parturition), PPD4 and before termination. Body weights of the female animals were additionally taken on gestational Days GD10 and 17 in order to give accurate treatment volumes but these data were not evaluated statistically.

- Food consumption measurement:
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g at least weekly (on the days of body weight measurements).

- Clinical pathology:
All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. For terminal blood sampling three samples were taken from each selected animal (5 males and 5 females/group), one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

- Haematology and blood clotting times: Daily or detailed weekly observation; For details on evaluated parameters see under 'any other information on material and methods incl. tables'

- Clinical chemistry: Daily or detailed weekly observation; For details on evaluated parameters see under 'any other information on material and methods incl. tables'

- Urinalysis:
Urine samples were collected for 16 hours during an overnight period of food deprivation during the last week of the study (Day 27-28 for males and PPD4-5 for female animals, respectively) from each animal by placing the animals in metabolic cages. The evaluation of the urine samples were performed by observation (e.g. appearance, colour) and test strips. For details on evaluated parameters see under 'any other information on material and methods incl. tables'

Sacrifice and pathology:

Pathology
- Gross necropsy was performed on all animals, irrespective of the date of death. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded in the females as applicable.

Organ weight measurements
- At the time of termination, body weight and the weight of the following organs from all euthanized adult animals were determined.
- With a precision of 0.01 g: brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Paired organs were weighed together. Individual and/or paired absolute organ weights were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.

Tissue preservation and microscopic evaluation.
- The weighed organs and all organs showing macroscopic lesions were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. All other organs in 10% buffered formalin solution. The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Detailed histological examination was performed on all retained organs in the control and High dose groups and any macroscopic findings (abnormalities) observed in all animals. (Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.).

For details on evaluated tissues and organs see under 'any other information on material and methods incl. tables'

Statistics:

Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, or using the software PROVANTIS v.9, as appropriate. Group means and standard deviations were calculated from numerical data obtained in the study.
The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) by an appropriate statistical method (Bartlett, ANOVA/ANCOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests, T-test, Wilcoxon test, Chi2 test). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA/ANCOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test or Kruskal-Wallis test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible. For SMART evaluation, T-test was used.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Terminally euthanised animals:
- Male animals: no clinical signs were detected in the Control, Low and Mid dose groups during the study. In the High (1000 mg/kg bw/day) dose group liquid faeces, noisy respiration and piloerection were occasionally present up to three animals during the treatment. These observations were considered to be treatment related.
- Female animals: thin fur/alopecia was observed in all groups, peaking at the end of the experiment, but is not considered to be related to test substance. No clearly adverse effects of the test substance related were noted in the Low dose groups during the study. In the Mid (300 mg/kg bw/day) dose group, piloerection and noisy respiration was observed sporadically in just 1 or 2 females. These non-specific observations seen sporadically at a low incidence is not considered to be a clear sign of an adverse effect of the test substance. In the High (1000 mg/kg bw/day) dose group, the non-specific signs of piloerection and noisy respiration was seen in a small number of male and the majority of females. Hunched back was also present in up to three female animals, which may be treatment related. A 1 to 2 cm wide crust was seen on the skin for five days in one animal, yellow coloured discharge in one animal, and discharge from vagina in one animal was observed during the treatment; these findings are of uncertain relationship with treatment.

Found dead and preterminally euthanized animals:
No common symptoms were found prior unanticipated deaths. The male animal showed slightly noisy, moderately laboured respiration one day before it was found dead. Two females showed no clinical signs before the time of death. Piloerection and slight to moderate noisy respiration and/or laboured respiration with hunched back was present in the rest of the animals, with red coloured discharge or distended abdomen prior dead or euthanasia. These symptoms were considered to be test substance related.

Excluded animals
Four females (two from the Control Dose group and two from the Mid (300 mg/kg bw/day) Dose group were excluded from certain statistical analysis (such as body weight) after being found to be sperm positive, but delivered no offspring despite detectable corpora lutea. Since body weight is affected by pregnancy, it is valid to exclude the non-pregnant animals from this data analysis. Sporadic occurrence of noisy respiration in one Control group and the two Mid dose animals, laboured respiration and piloerection in one Mid dose animal was observed, but these observations were not considered to be test item related.

Mortality:

mortality observed, treatment-related

Description (incidence):

A test substance related mortality (approximately 15% mortality) was observed in the High dose group (1000 mg/kg bw/day). One male (Day 16) and 4 females (Days 3, 6, 18 and 22 ) of the High dose group were found dead. In addition, one female (Day 17) was preterminally euthanized.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance related effects were noted on the mean body weight and body weight gain values following daily administration of test substance at dose levels up to and including 1000 mg/kg bw/day

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance related differences in the mean daily food consumption in any test substance treated group when compared to the controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to the controls, there were no differences that could be considered toxicologically significant in the Low and Mid dose (100 and 300 mg/kg bw/day, respectively) groups. The High (1000 mg/kg bw/day) males only had a slightly but significantly (p<0.05) lower Mean Cell Haemoglobin Concentratin (MCHC). The female High dose group had a slightly but significantly (p<0.05) longer Activated Partial Thromboplastin Time (APTT). However the data from all animals was in the normal range and these small differences were not consistent between sexes and were not considered to be an adverse effect of treatment.
For other parameters, evaluation of the mean and individual results in comparison with the control data did not reveal any test-substance related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range and of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the animals evaluated at termination (on Day 28 in males and on PPD5 in females), there were no clearly toxicologically significant changes or adverse effects on the animal serum chemistry that could be ascribed to the test substance administration in the conditions of this study. Urea and chloride concentrations were statistically significantly higher in the male High (1000 mg/kg bw/day) dose group compared to the Control (p<0.05 and p<0.01, respectively). The female High dose group had statistically significantly (p<0.05) lower phosphorus values. However the data for these 3 parameters from all animals was in the normal range and these small differences were not consistent between sexes and were not considered to be an adverse effect of treatment.
Some differences in the Low and Mid treated groups also attained statistical significance; however, there was no dose or gender response or the values were within the physiological ranges. For this reason, these variations were not considered toxicologically significant or related to treatment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

A dose dependent decrease of pH was observed in both sexes, reaching statistical significant difference compared to the control value at the High (1000 mg/kg bw/day) dose males (p<0.05) and females (p<0.01). In the High dose only, slight but statistical significance differences were recorded with lower pH and higher urine gravity in both sexes (p<0.01) and lower urine volume in makes only (p<0.05). However, all values were in the normal control range, these findings were regarded as minor variations and were not considered to indicate an adverse effect of the test substance.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidney weights were slightly higher than control in both sexes at the High dose (by 8% in males and by approximately 10% in females). This was statistically significant both in terms of absolute weight or when adjusted for body or brain weights. There were also slight differences in urine analysis at the High dose level in both sexes, so the small organ weight differences may reflect an adaptive or functional difference. A similar statistically significant difference was seen in female kidney weights in the Mid dose group but the data was all in the normal range. Therefore it was not considered to be a clear effect of the test substance. At the Mid and Low dose levels, it is considered that minor statistical differences did not reflect an effect of the test susbtance. In the absence of any histopathological or blood chemistry changes, there is no evidence of an adverse effect in the kidneys at any dose level.
In females, increased liver weights were seen in the High dose group only (by 12% absolute weight). Increased liver weights were not seen in the males of the High dose group. These liver weight changes correlate well with the centrilobular hepatocellular hypertrophy observed in High (1000 mg/kg bw/day) dose female animals.
There were no other toxicologically significant differences among groups in the weights of other organs measured when compared to controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Found dead animals: Small spleen in 2 out of 4 found dead females and small thymus in 1 out of 4 female corresponded with findings noted during microscopic examination.
- Preterminal euthanasia: Small spleen and thymus, pale foci of the left lobe in the liver were observed at necropsy and were related to the treatment. Dilatation (with gas) of the oesophagus, stomach, duodenum, jejunum, ileum, caecum and colon, and red/clear material at the perinasal fur did not have relationship to the treatment.
- Terminal animals: No treatment-related macroscopic findings were observed up to the dose level of 1000 mg/kg bw/day.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Neurological:
There were no toxicologically significant changes in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test groups.
There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. Grip strengths of the forelimb were significantly (p <0.05) lower in Mid dose (300 mg/kg bw/day) females, but without dose dependency and similar results for the hindlimbs or during the modified Irwin test, this variety was not considered to be a test item related effect.
The total travelled distance in the High dose group and the shape of the curve of activity in 5 minute periods over one hour, were comparable to the control groups for both sexes; all data was within the normal expected range. Sporadic statistical significant differences were of no toxicological relevance. There was no test item related effect on locomotor activity in either sex.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Found dead animals:
Treatment-related slight centrilobular hepatocellular hypertrophy of the liver was observed in 1 out of 4 female. Various microscopic findings induced by stress due to treatment such as slight decrease of white pulp size/cellularity in the spleen and moderate decrease size/cellularity of the thymic cortex (correlated with gross changes), slight decrease of myeloid cellularity in the femoral and sternal bone marrows, slight diffuse vacuolation of the adrenal’s medulla were present in this found dead female. In addition, moderate decrease of white pulp size/cellularity in the spleen was also recorded in one other female rat. There were no findings that clearly indicated a cause of death.

- Preterminal euthanasia:
Treatment-related slight centrilobular hepatocellular hypertrophy of the liver was seen by light microscopy and visualized at necropsy (pale foci). The histopathological findings corresponded with stress-related responses and ascribed to the treatment included moderate decrease of white pulp size/cellularity in the spleen, moderate decrease size/cellularity of the thymic cortex and, correlated with gross changes. Additionally, decrease myeloid cellularity in the femoral (moderate severity) and sternal (slight severity) bone marrows, slight diffuse vacuolation of the adrenal’s medulla, were noted by light microscopy. There were no findings that clearly indicated a cause of a moribund state.
Other changes were regarded as incidental.

- Terminal animals:
Treatment-related centrilobular hepatocellular hypertrophy of the liver was seen at a dose level 1000 mg/kg bw/day. Minimal hypertrophy altered liver in 1 out of 5 selected male and minimal/slight hypertrophy in 4 out of 5 selected females. This change was considered to be an adaptive non-adverse response to the administration of the test substance.
Other changes were incidental or a common background.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: for systemic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the rat NOAEL for systemic effects was established at 300 mg/kg bw/day for females and males.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the test substance according to OECD guideline 422. The general systemic toxic potential of the substance to Wistar rats was assessed by daily oral administration (gavage). Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating), and females for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 days post-partum dosing). Three groups each comprising at least 12 male and 12 female rats received the test substance at doses of 0, 100, 300 or 1000 mg/kg bw/day. Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives. A detailed histological examination was performed on the selected list of retained organs in the control and High dose groups. Daily administration of the test substance at dose levels of 100 or 300 mg/kg bw/day did not result in adverse changes in clinical signs, neurological assessment, body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters. Test substance related mortality (approximately 15% mortality) was observed in the High dose group (1000 mg/kg/d) and clinical adverse effects were present in High dose females. There were no effects on surviving animals on body weight or clinical pathology. Test substance related liver changes were observed in the female High dose group (1000 mg/kg bw/day) with increased weight (approximately 12%) and centrilobular hepatocellular hypertrophy. However, this observation was considered to be an adaptive change and not an adverse effect of treatment. Test substance related kidney weight changes were observed in the High dose group of both sexes (by up to about 10%), but in the absence of any histological changes, this too was considered to be an adaptive response. Under the study conditions, the rat NOAEL for systemic effects was established at 300 mg/kg bw/day for females and males (Hargitai, 2016).