Hexane, 1,6-diisocyanato-, homopolymer, 2-hydroxyethyl acrylate- and propylene glycol monoacrylate-blocked

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 26, 2016 to January 02, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 26, 2016 to January 2, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

did not affect outcome/integrity of the study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

did not affect outcome/integrity of the study

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

did not affect outcome/integrity of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

Test system Rat: Crl:WI(Han) (outbred, SPF-Quality).
Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
Rationale: This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age: Approximately 10-12 weeks;
Number of animals: 40 females and 40 males;
Acclimatization: At least 5 d prior to start of treatment;
Health inspection: At least upon receipt of the animals;
Randomization: Before initiation of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean;
Identification: Earmark and tattoo.

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/h, and a 12-h light/12-h dark cycle. The light/dark cycle was interrupted for study related activities.

Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of approximately 2.5 h. There has been provided a free access to tap-water.

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Vehicle:

propylene glycol

Details on oral exposure:

Based on the results of a dose range finding study in which dose levels of 500 and 1000 mg/kg bw were tested (17 and 10 d, respectively), no dose limiting effects were seen, the dose levels for the main study were therefore selected to be 100, 300 and 1000 mg/kg bw/d.

Analytical verification of doses or concentrations:

yes

Remarks:

LC-MS/MS

Details on analytical verification of doses or concentrations:

No test substance was detected in the control group formulation. The concentrations analyzed in the formulations of groups 2, 3 and 4 (100, 300 and 1000 mg/kg bw/d) were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and Group 4 prepared were homogeneous (i.e. coefficient of variation ≤ 10%).
Formulations at the entire range were stable when stored at room temperature protected from light for at least 5 h (i.e. relative difference ≤ 10%).
The test substance was stable in propylene glycol over a storage period of at least 86 d at a temperature ≤ -70°C.
A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Duration of treatment / exposure:

- Males were dosed for 29 d i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
- Females that delivered were exposed for 40-45 d i.e. 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 4 d after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were exposed for 40 or 42 d.

Frequency of treatment:

Once daily for 7 d per week

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

The test substance, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups (100, 300 and 1000 mg/kg bw/day) of the test substance were tested, each consisting of 10 males and 10 females.

Positive control:

No positive control was used.

Observations and examinations performed and frequency:

Mortality/Viability: At least twice daily.

Clinical signs: At least once daily from start of treatment up to the day prior to necropsy, detailed clinical observations were made for all animals, at least 1 h (± 30 min) after dosing (based on the peak period of anticipated effects after treatment). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Functional Observations: The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group:
• hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength, recorded as the mean of three measurements per animal.
• locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system.
Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
The selected males were tested during Week 4 of treatment and the selected females were tested during the lactation period from Day 4 of lactation onwards (all before blood sampling). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).

Body weights: Males and females were weighed on the first day of exposure (prior to first exposure) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

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

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

Haematology and clinical biochemistry parameters were evaluated. Samples collection:
Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The selected animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.

Sacrifice and pathology:

- Necropsy: 5 animals/sex/group - Males following completion of the mating period, females which delivered, females which failed to deliver (females with evidence of mating).

- Gross lesions of all animals were recorded (all dose groups).

- Terminal body weights were recorded from all males and the selected 5 females/group. The following organ weights were recorded from the following animals on the scheduled day of necropsy: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid (including parathyroid), uterus (including cervix).

- Pathology examinations:
The numbers of corpora lutea and former implantation sites were recorded for all paired females. Samples of the following tissues and organs were collected and fixed in 10% buffered formalin for further histophatological examinations: ovaries, adrenal glands, pancreas, aorta, peyer's patches, brain (cerebellum, mid-brain, cortex), pituitary gland, caecum, preputial gland, cervix, prostate gland, clitoral gland rectum, colon, salivary glands (mandibular, sublingual), coagulation gland sciatic nerve, duodenum seminal vesicles, epididymide skeletal muscle, eyes, skin, female mammary gland area, spinal cord (cervical, midthoracic, lumbar), femur including joint, spleen, heart, sternum with bone marrow, ileum, stomach, jejunum, testes, epididymides, kidneys, thymus, lacrimal gland, exorbital, thyroid including parathyroid, larynx, tongue, liver, trachea, lung, urinary bladder.

Statistics:

Statistical Analyses:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations.
- Salivation occurred after dosing in all males and females treated with the test substance, generally starting in the first week of treatment. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing), and may be related to the irritancy of the test substance. In addition, most treated rats showed slight lethargy after dosing on the first day of the treatment period. This clinical sign was considered not to be toxicologically relevant because it was no longer observed during the remainder of the treatment period. Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain was higher during the first two weeks of the treatment period in males and females treated at 1000 mg/kg bw/d. Thereafter, growth rate at 1000 mg/kg bw/d was comparable to that in controls (total weight gain in males remained above control levels until the end of the study). Mean body weights of 1000 mg/kg bw/d males were close to control values (about 3% higher) while 1000 mg/kg bw/d females had statistically significantly higher body weights on Days 1 and 4 of lactation (relative difference 7%). As the changes in body weight (gain) were slight, they were considered to be of no toxicological significance. Body weight and body weight gain were not affected by treatment at 100 or 300 mg/kg bw/d.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was generally similar between treated and control animals. The slightly higher values noted in females at 300 and/or 1000 mg/kg bw/d at the end of the post-coitum period (statistically significant for absolute values) and during lactation (not statistically significant) were consistent with the slightly higher body weights of these females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological parameters were not affected by treatment. One female at 1000 mg/kg bw/d had outlying values for platelets (higher) and red blood cell parameters (lower number of red blood cells and haemoglobin, with compensatory changes consisting of higher reticulocytes, red blood cell distribution width (RDW) and meancorpuscular volume (MCV), and increased extramedullary hematopoiesis in the spleen and liver). The reduction in red blood cells and haemoglobin could be due to blood loss at parturition. As these changes were observed in only a single animal and recovery was ongoing, they were considered not to reflect adverse effects of the test substance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in clinical biochemistry parameters of treated rats. The variations noted in clinical biochemistry values, including those achieving statistical significance, were unrelated to treatment or not toxicologically relevant due to the slight magnitude of the difference from controls (values in treated rats remained within normal limits) and/or the direction of the change.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Test substance-related higher liver weights (absolute and/or relative to body weight) were noted at 1000 mg/kg bw/d in both sexes. Relative differences from the control group are noted below.
Males dose level / relative to body weigh liver growth:
100 mg/kg bw: 3
300 mg/kg bw: 1
1000 mg/kg bw: 8
Females dose level, relative to body weight:
100 mg/kg bw: -5
300 mg/kg bw: 2
1000 mg/kg bw: 6 (P > 0.05)
This change was regarded as non-adverse based on its small magnitude and the absence of corroborative changes indicative of hepatotoxicity.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopic observations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, there were no toxicologically relevant changes in the in-life parameters up to the highest dose of 1000 mg/kg bw/d and the repeat dose NOAEL of the substance was determined to be >1000 mg/kg bw/d in rats.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the substance according to OECD Guideline 422, in compliance with GLP. The test substance, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three test substance groups (100, 300 and 1000 mg/kg bw/d) were tested, each consisting of 10 males and 10 females. Males were dosed for 29 d, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 40 -45 d, i.e. 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 4 d after delivery up to and including the day before scheduled necropsy. The following observations and examinations were performed: mortality, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), gross pathology (at termination), organ weights, and histopathology on a selection of tissues. A few non-adverse, treatment-related findings were observed during the in-life phase in both sexes. Males and females treated at 1000 mg/kg bw/d had statistically significant higher liver weights (absolute and/or relative to body weight). This change was regarded as non-adverse due to its small magnitude and the absence of corroborative changes indicative of hepatotoxicity. No treatment-related or toxicologically relevant changes were recorded in clinical pathology parameters, remaining organ weights, or at macroscopic and microscopic examinations. Under the study conditions, there were no toxicologically relevant changes in the treated rats up to the highest tested dose of 1000 mg/kg bw/d and the repeat dose NOAEL of the substance was determined to be >1000 mg/kg bw/d (de Raaf - Beekhuijzen, 2017).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

The study integrity was not adversely affected by the deviations.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviations.

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviations.

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviations.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexane, 1,6-diisocyanato-, homopolymer, 2-hydroxyethyl acrylate- and propylene glycol monoacrylate-blocked

EC Number:

806-984-5

Cas Number:

1392411-89-0

IUPAC Name:

Hexane, 1,6-diisocyanato-, homopolymer, 2-hydroxyethyl acrylate- and propylene glycol monoacrylate-blocked

Test material form:

liquid: viscous

Details on test material:

Name: Ebecryl 4738, Batch no.: DR0004393

Specific details on test material used for the study:

Batch: DR0004393; Purity/Composition: UVCB; Appearance: Clear to slightly yellow viscous liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

Test system Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and nonpregnant females and untreated animals were used at initiation of the study
Source: P0 Charles River Deutschland, Sulzfeld, Germany
Rationale: This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effect of reproductive toxicants

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age: at start P0-treatment Approximately 10-12 weeks;
Number of P0-animals: 40 females and 40 males;
Acclimatization P0: At least 5 d prior to start of treatment;
Health inspection P0: At least upon receipt of the animals;
Randomization P0: Before initiation of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean;
Identification P0: Earmark and tattoo.

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12 h light/12 h dark cycle. The light/dark cycle was interrupted for study related activities.

General: Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of approximately 2.5 h. There has been provided a free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of approximately 2.5 h.

Pre-mating: Animals were housed in groups of 5 animals of the same sex in Macrolon plastic cages (MIV type, height 18 cm). Post-mating: same type of cage used, males and females kept separetely. Mating: same type of cage used, animals kept in one-to-one-basis.
Lactation: Females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 min.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Details on mating procedure:

Following a minimum of 14 d of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 d was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Detection of mating was not confirmed in first instance for four animals which delivered live offspring. The mating date of these animals was determined by rechecking of the vaginal lavages. This day was designated Day 0 post-coitum. Detection of mating was not confirmed for one animal which delivered live offspring. The mating date of this animal was estimated at 21 d prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Remarks:

LC-MS/MS

Details on analytical verification of doses or concentrations:

No test substance was detected in the control group formulation. The concentrations analyzed in the formulations of groups 2, 3 and 4 (dosages to each group respectively: 100, 300, 1000 mg/kg bw) were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and Group 4 prepared were homogeneous (i.e. coefficient of variation ≤ 10%).
Formulations at the entire range were stable when stored at room temperature protected from light for at least 5 h (i.e. relative difference ≤ 10%).
The test substance was stable in propylene glycol over a storage period of at least 86 d at a temperature ≤ -70°C.
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Duration of treatment / exposure:

- Males were dosed for 29 d, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
- Females that delivered were exposed for 40-45 d (most females) or 53 d (one female), i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 4 d after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were exposed for 40 or 42 d. Routinely, females that are littering are left undisturbed.

Frequency of treatment:

Once daily for 7 d per week, approximately the same time each day with a maximum of 6 h difference between the earliest and latest dose.

Details on study schedule:

Experimental starting date: 26 September 2016 (Randomization Dose Range Finder);
Start treatment: 10 November 2016;
Start mating: 24 November 2016;
Blood sampling: 9 December 2016 (selected males);
21 - 24 December 2016 (selected females);
Delivery of litters: (PND 1) 17 – 20 and 28 December 2016;
Necropsy: 09 December 2016 (males), 20 - 25 December 2016 and 02 January 2017 (females and pups);
Experimental completion date: 02 January 2017 (end of in-life phase).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group 1

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

The test substance, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups (100 mg/kg bw/day, 300 mg/kg bw/day, 1000 mg/kg bw/day of the test substance were tested, each consisting of 10 males and 10 females.

Positive control:

no positive control

Examinations

Parental animals: Observations and examinations:

General systemic data: mortality, clinical signs, body weight, food consumption, water consumption, functional observations (hearing ability, pupillary reflex, and static righting reflex, fore- and hind-limb grip strength, locomotor activity). For details on systemic effects, please refer to section RDT: oral.

General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females were examined for evidence of premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Slides of the testes of the selected 5 males of Groups 1 and 4 and of all males that failed to sire twere examined for the staging of spermatogenesis.

Litter observations:

Each litter was examined to determine the following, if practically possible:
Mortality / Viability;
Clinical signs: at least once daily for all animals;
Body weights: live pups were weighed on PND 1 and 4.
Sex: determined for all pups on PND 1 and 4. Sex ratio (% male pups / % female pups) was calculated per group.

Postmortem examinations (parental animals):

Necropsy: Selected 5 animals/sex/group - Males Following completion of the mating period, females which delivered, females which failed to deliver and females with evidence of mating.

Pathology examinations:
The numbers of corpora lutea and former implantation sites were recorded for all paired females. Samples of the following tissues and organs were collected and fixed in 10% buffered formalin for further histophatological examinations: ovaries, adrenal glands, pancreas, aorta, peyer's patches, brain (cerebellum, mid-brain, cortex), pituitary gland, caecum, preputial gland, cervix, prostate gland, clitoral gland rectum, colon, salivary glands (mandibular, sublingual), coagulation gland sciatic nerve, duodenum seminal vesicles, epididymide skeletal muscle, eyes, skin, female mammary gland area, spinal cord (cervical, midthoracic, lumbar), femur including joint, spleen, heart, sternum with bone marrow, ileum, stomach, jejunum, testes, epididymides, kidneys, thymus, lacrimal gland, exorbital, thyroid including parathyroid, larynx, tongue, liver,trachea, lung, urinary bladder.

Terminal body weights were recorded from all males and the selected 5 females/group. The following organ weights were recorded from the following animals on the scheduled day of necropsy: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid (including parathyroid), uterus (including cervix).

For all remaining animals: epididymides, testes examined.

Histophatology examinations:
The following slides were examined:
• The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4
• Additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males that failed to sire were examined for the staging of spermatogenesis.
• All gross lesions of all animals (all dose groups).
• The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5 or 6 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis non-parametric ANOVA test was applied to motor activity data to determine intergroup differences.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%)
Fertility index (%)
Conception index (%)
Gestation index (%)
Duration of gestation

Offspring viability indices:

Percentage live males at First Litter Check
Percentage live females at First Litter Check
Viability index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The spermatogenic staging profiles were normal for all males examined.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

- Precoital time was not affected by treatment.
- Fertility and conception index were not affected by treatment. One female of the control group, one female dosed at 100 mg/kg bw/day and one female dosed at 300 mg/kg bw/day were not pregnant. These incidental non-pregnancies, which occurred in absence of related histopathology changes in reproductive organs or a dose-related trend, were considered to be unrelated to treatment.
- Gestation index and duration of gestation were not affected by treatment.
- Numbers of corpora lutea and implantation sites were not affected by treatment. For one female (100 mg/kg bw/day) the number of pups was slightly higher than the number of corpora lutea and implantation sites. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 5 of lactation.
- There were no morphological findings in the reproductive organs of either sex which could be attributed to the test substance.
- No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related systemic adverse effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related adverse effects on reproduction

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed remained within the range considered normal for pups of this age and showed no dose-related trend. They were therefore considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- At first litter check, one pup of the control goup and one pup at 1000 mg/kg bw/day were found dead. This incidental pup mortality was unrelated to treatment with the test substance. The number of living pups at first litter check was unaffected by treatment.
Viability index (number of offspring surviving until planned necropsy as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices across the groups were 95-100%.
- During lactation (mostly on PND 2), two pups of the control group, two pups at 100 mg/kg bw/day, and four pups at 300 mg/kg bw/day went missing. These missing pups were most likely cannibalized. No toxicological relevance was attributed to this post-natal mortality since the incidence of missing pups showed no dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes were noted in body weights of pups. Compared to controls, male and female pups at 1000 mg/kg bw/day appeared to have slightly higher body weights at PND 1 and 4 with slightly higher body weight gain between PND 1-4. The differences were not statistically significant and remained in the normal range for pups of this age. Therefore, this finding was considered not to be toxicologically relevant.

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:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were observed among pups that were considered to be related to treatment. The nature and incidence of the few findings observed remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were observed among pups that were considered to be related to treatment.

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related adverse effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the oral NOAEL of the substance for reproductive/developmental toxicity in rats was determined to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to determine the toxicity of the substance on reproduction/development of rats according to OECD Guideline 422, OECD Guideline 421, OPPTS Guideline 870.3650 and OPPTS Guideline 870.3550, in compliance with GLP. The study focused on gonadal observations and reproduction/developmental effects. The details on systemic effects are summarised in repeated dose toxicity (oral) section 7.5.3. of IUCLID dataset. The test substance, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups (0, 100, 300 and 1000 mg/kg bw/day) of the test substance were tested, each consisting of 10 males and 10 females. Males were treated for 29 d, i.e. 2 weeks prior to mating, during mating, and until scheduled necropsy. Females were treated for 40 -45 d, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and at least 4 d of lactation. For parental generation, the following reproduction parameters were examined: mating, fertility and conception indices, precoital time, numbers of corpora lutea and implantation sites, gestation index and duration, parturition, and maternal care. The offspring was evaluated during early lactation period for mortality, clinical signs, body weights, and gross pathology. The sex ratio was also recorded. No treatment-related changes were observed in any of the systemic, reproductive or developmental parameters investigated, either in parents or pups. Indeed, precoital time, fertility, conception index, gestation index and duration, numbers of corpora lutea and implantation sites were not affected by treatment. Moreover there were no morphological findings in the reproductive organs of either sex which could be attributed to the test substance. The test substance did not induce any effects in the early development of the pups. Under the study conditions, the oral NOAEL of the substance for reproductive/developmental toxicity in rats was determined to be 1000 mg/kg bw/day (de Raaf - Beekhuijzen, 2017).