1-Octanol, reaction products with phosphorus oxide (P2O5), potassium salts

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31-July-2018 to 28-January-2019

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:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31-July-2018 to 28-January-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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)

Version / remarks:

2016

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Purity: UVCB; solid matter 41.4%; Correct for % solid matter

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. 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 effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 14-15 weeks old; Study males were slightly older than the standard age (14-15 weeks instead of 10-12 weeks) at initiation of dosing. As the test item was dosed undiluted, slightly older males were used to increase the actual dose volume.
- Weight at study initiation: Males: 332 to 393 g; Females: 217 to 256 g
- Housing:
On arrival and following the pretest (females only) and pre-mating period, animals were group housed, up to 5 animals of the same sex and same dosing group together. During the mating phase, males and females were cohabitated on a 1:1 basis. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed. During the lactation phase, females were housed with pups, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22°C
- Humidity (%): 42 to 73%.
- Air changes (per hr): 10 or more per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

unchanged (no vehicle)

Remarks:

Test-item treated animals were received undiluted test item and consequently, no vehicle was used.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution as received. The test item has 41.4% solid matter and as such test item concentration was corrected for % solid matter and specific gravity (factor: 1.094).

Analytical verification of doses or concentrations:

no

Remarks:

The test item was used as received from the Sponsor; therefore, samples for dose formulation analysis were not collected by the Test Facility.

Details on analytical verification of doses or concentrations:

The test item was used as received from the Sponsor; therefore, samples for dose formulation analysis were not collected by the Test Facility.

Duration of treatment / exposure:

A minimum of 28 days. Males were treated for 29 days, i.e. 14 days prior to mating, during mating and up to and including the day before scheduled necropsy. Females that delivered were treated for 50-55 (most females) or 63 days (one female at 150 mg/kg), i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 53 or 42 days, respectively.

Frequency of treatment:

Once daily, 7 days per week. Female nos. 50 (Control), 58 and 60 (Group 2), were not dosed on one occasion as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Dose selection was based on the results of the dose range finder. In the range finder dose-limiting effects were observed after short-term treatment (10 days) at 1000 mg/kg [i.e. increase in mean relative liver weight of 39% compared to historical control mean, combined with body weight loss in one female, hunched posture and piloerection in all females (clinical observations) and irregular surface of the forestomach in all females (macroscopic examinations)].
- Fasting period before blood sampling for clinical biochemistry: Blood of F0-animals (except for animal nos. 17 and 66 which were sacrificed in extremis and female no. 63 with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis. F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

Positive control:

Not examined

Observations and examinations performed and frequency:

Five animals/sex/group were selected for functional tests, clinical pathology, collection of full list of organs/tissues at macroscopic examination, organ weights (full list) and histopathology (full list).

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: weighed on the first day of treatment (prior to dosing), 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, 4, 7, and 13. Terminal body weights were recorded on the day of scheduled necropsy (fasted for males and non-fasted for females).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not feeding study. Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not drinking water study. Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day of scheduled necropsy
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 8-11).
- Dose groups that were examined: All groups
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex, static righting reflex), grip strength, motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (all animals were examined for necropsy).

HISTOPATHOLOGY: Yes
Based on treatment-related changes in tissues, the stomach of selected males and females of low and mid-dose groups (5/sex/group) were examined.

Male no. 17 (Group 2) and female no. 66 (Group 3) were euthanized as per Test Facility SOPs. These animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained but not weighed.

Other examinations:

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio, live litter size and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 14-16 pups and macroscopy).

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Slight salivation after dosing, considered a physiological response to the irritant test item rather than a sign of systemic toxicity, occurred among animals treated at 150 or 500 mg/kg in a dose-related manner. Rales occurred transiently in several treated males (at all dose levels, without a dose-related trend). This respiratory symptom was attributed to the oral gavage administration of the irritant test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two treated animals (a low-dose male and a mid-dose female) were sacrificed prematurely for humane reasons. These premature deaths were regarded to be related to the oral gavage administration of the irritant test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A finding of note was the weight loss of one high-dose male (no. 39). This male lost 11% of its initial weight during treatment weeks 1-3 (no further loss in the next week). This was accompanied by hunched posture and laboured respiration on Days 7-11/10. Macroscopic and microscopic examination revealed no obvious cause of the weight loss (no. 39 showed similar gastric changes as most other high-dose males which grew normally; microscopy was limited to the stomach). The weight loss of no. 39 was considered not to reflect an adverse effect of the test item on body weight (gain) since such weight loss occurred in only a single (surviving) animal.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences between treated rats and controls were limited to a lower mean corpuscular haemoglobin (MCH) value in high-dose (500 mg/kg) males (relative difference: -4%). As main red blood cell parameters (haemoglobin concentration, number of red blood cells) of high-dose males were not affected, the lower MCH was considered not to reflect an adverse effect of the test item on red blood cells.
The abnormal white blood cell values noted in low-dose female no. 59 (higher numbers of neutrophils and total white blood cells, lower number of lymphocytes) were likely to be related to a thymoma (which was unrelated to treatment).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes in clinical pathology parameters consisted of increases in cholesterol and bile acids and a decrease in total protein in males treated at 500 mg/kg. In the absence of associated adverse anatomic pathology alterations, these changes were regarded as non-adverse.

Urinalysis findings:

not examined

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):

Organ weight changes at 500 mg/kg consisted of increased absolute and relative weights of the liver in both sexes and increased relative weights of the thymus and kidneys in males. These organ weight changes were regarded as non-adverse as they were not associated with histopathological alterations.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic findings were present in the stomach of one male at 150 mg/kg and most males 500 mg/kg. These findings consisted of an irregular surface of the forestomach (in 1/10 and 8/10 males at 150 and 500 mg/kg, respectively), thickened limiting ridge and/or glandular stomach (8/10 males at 500 mg/kg), and red focus/foci in the glandular stomach (5/10 males at 500 mg/kg).
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The microscopic correlates were inflammation and erosions in the forestomach and glandular stomach, hyperkeratosis (and a few males with squamous cell hyperplasia) in the forestomach, and submucosal edema and hemorrhages in the glandular stomach.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Mean serum level of T4 in males treated at 500 mg/kg was 16% lower than the concurrent control mean. However, as statistical significance was not achieved and mean T4 value in 500 mg/kg treated males remained within the historical control range, this change was attributed to biological variation.

Details on results:

Treated males showed many inflammatory and correlating findings as described below.
Forestomach (non-glandular):
- Inflammation (mononuclear, epithelial and/or lymphogranulocytic, subepithelial) up to moderate degree starting at 150 mg/kg.
- Hyperkeratosis up to moderate degree starting at 50 mg/kg.
- Squamous cell hyperplasia up to slight degree at 500 mg/kg.
- Moderate erosion in a single male treated at 150 mg/kg.
- Increased vacuolation up to moderate degree at the limiting ridge starting at 150 mg/kg.
Glandular stomach:
- Increased inflammation (submucosal, eosinophilic or lymphogranulocytic) up to moderate degree starting at 150 mg/kg.
- Slight erosion in a single male at 500 mg/kg.
- Hemorrhages up to slight degree at 500 mg/kg.
- Hypertrophy of mucous cells up to slight degree starting at 50 mg/kg.
- Eosinophilic globules up to slight degree at 500 mg/kg.
- Submucosal edema up to moderate degree starting at 50 mg/kg.

Key result

Dose descriptor:

NOAEL

Remarks:

parental systemic

Effect level:

500 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

other: clinical pathology - non-adverse

Key result

Dose descriptor:

NOAEL

Remarks:

Parental local

Effect level:

50 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

The gastric findings in males at 50 mg/kg (minimal hyperkeratosis at the limiting ridge, and hypertrophy of mucous cells or submucosal edema in the glandular stomach) were considered to be non-adverse based on minimal severity and lack of degenerative/proliferative changes.

Test item-related changes in clinical pathology parameters consisted of increases in cholesterol and bile acids and a decrease in total protein in males treated at 500 mg/kg. In the absence of associated adverse anatomic pathology alterations, these changes were regarded as non-adverse.

Conclusions:

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect levels (NOAELs) of the test substance were established:
Parental systemic NOAEL: at least 500 mg/kg.
Parental local NOAEL: 50 mg/kg, based on adverse histopathological changes in the stomach (glandular and non-glandular) in males starting at 150 mg/kg.

Executive summary:

In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, Wistar Han rats were treated with undiluted test substance by daily oral gavage at dose levels of 50, 150 and 500 mg solid matter/kg (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received water (Elix). Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (mostly for 50-55 days). A female without offspring (non-mated) was treated for 53 days and a female which had a total litter loss was treated for 42 days.

 

Two treated animals (a low-dose male and a mid-dose female) were sacrificed prematurely for humane reasons. These premature deaths were regarded to be related to the oral gavage administration of the irritant test item.

 

Slight salivation after dosing, considered a physiological response to the irritant test item rather than a sign of systemic toxicity, occurred among animals treated at 150 or 500 mg/kg in a dose-related manner. Rales occurred transiently in several treated males (at all dose levels, without a dose-related trend). This respiratory symptom was attributed to the oral gavage administration of the irritant test item.

 

Male rats showed morphological changes in the stomach (glandular and non-glandular) which were regarded as local effects resulting from the irritant properties of the test item. Although gastric changes occurred at all dose levels, in a dose-related manner, the combination of the severity and the degenerative/proliferative nature of the gastric lesions at 150 and 500 mg/kg was considered to be adverse. Main findings in the forestomach consisted of inflammation (epithelial mononuclear infiltrates and subepithelial lymphogranulocytic infiltrates), hyperkeratosis and squamous cell hyperplasia. Main findings in the glandular stomach were increased inflammation (submucosal, eosinophilic or lymphogranulocytic), mucosal hemorrhage, hypertrophy of mucous cells, eosinophilic globules and submucosal edema). Erosion in the glandular or non-glandular stomach and increased epithelial vacuolation at the limiting ridge were observed incidentally. Macroscopic changes consisted of an irregular surface of the forestomach (in one 150 mg/kg male and most 500 mg/kg males), thickened limiting ridge and/or glandular stomach (most 500 mg/kg males), and red focus/foci in the glandular stomach (half of the 500 mg/kg males). The gastric findings in males at 50 mg/kg (minimal hyperkeratosis at the limiting ridge, and hypertrophy of mucous cells or submucosal edema in the glandular stomach) were considered to be non-adverse based on minimal severity and lack of degenerative/proliferative changes.

 

Organ weight changes at 500 mg/kg consisted of increased absolute and relative weights of the liver in both sexes and increased relative weights of the thymus and kidneys in males. These organ weight changes were regarded as non-adverse as they were not associated with histopathological alterations.

 

Test item-related changes in clinical pathology parameters consisted of increases in cholesterol and bile acids and a decrease in total protein in males treated at 500 mg/kg. In the absence of associated adverse anatomic pathology alterations, these changes were regarded as non-adverse.

No reproduction or developmental toxicity was observed up to the highest dose level tested (500 mg/kg).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

Octan-1-ol, reaction products with diphosphorus pentaoxide, potassium salts

EC Number:

807-789-8

Cas Number:

111062-42-1

Molecular formula:

C8-H18-O.K.O5-P2

IUPAC Name:

Octan-1-ol, reaction products with diphosphorus pentaoxide, potassium salts

Specific details on test material used for the study:

- Purity: UVCB; solid matter 41.4%; Correct for % solid matter

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. 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 effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 14-15 weeks old; Study males were slightly older than the standard age (14-15 weeks instead of 10-12 weeks) at initiation of dosing. As the test item was dosed undiluted, slightly older males were used to increase the actual dose volume.
- Weight at study initiation: Males: 332 to 393 g; Females: 217 to 256 g
- Housing:
On arrival and following the pretest (females only) and pre-mating period, animals were group housed, up to 5 animals of the same sex and same dosing group together. During the mating phase, males and females were cohabitated on a 1:1 basis. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed. During the lactation phase, females were housed with pups, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22°C
- Humidity (%): 42 to 73%.
- Air changes (per hr): 10 or more per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Remarks on MMAD:

Not applicable (oral study)

Vehicle:

unchanged (no vehicle)

Remarks:

Test-item treated animals were received undiluted test item and consequently, no vehicle was used.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution as received. The test item has 41.4% solid matter and as such test item concentration was corrected for % solid matter and specific gravity (factor: 1.094).

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

Male no. 16 (50 mg/kg bw/day group) was initially paired with female no. 56 (she showed evidence of mating after one day). Subsequently, this male was paired with female no. 57 of the same group on Day 2 of the mating period to replace male no. 17 that was sacrificed for humane reasons on treatment Day 8.

A maximum of 14 days was allowed for mating, after which females which had not shown evidence of mating (nos. 57 and 65) were separated from their male.

Detection of mating was not confirmed in first instance for female no. 65. Evidence of mating was obtained by palpation and indirectly by delivery of a litter. Apparently, mating was overlooked in the initial assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating in this female. The mating date of this female was established at re-evaluation of the vaginal lavage (confirmed by evidence of sperm).

Analytical verification of doses or concentrations:

no

Remarks:

The test item was used as received from the Sponsor; therefore, samples for dose formulation analysis were not collected by the Test Facility.

Details on analytical verification of doses or concentrations:

The test item was used as received from the Sponsor; therefore, samples for dose formulation analysis were not collected by the Test Facility.

Duration of treatment / exposure:

A minimum of 28 days. Males were treated for 29 days, i.e. 14 days prior to mating, during mating and up to and including the day before scheduled necropsy. Females that delivered were treated for 50-55 (most females) or 63 days (one female at 150 mg/kg), i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 53 or 42 days, respectively.

Frequency of treatment:

Once daily, 7 days per week. Female nos. 50 (Control), 58 and 60 (50 mg/kg Group), were not dosed on one occasion as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.

Details on study schedule:

Females were allowed to litter normally. PND 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Dose selection was based on the results of the dose range finder. In the range finder dose-limiting effects were observed after short-term treatment (10 days) at 1000 mg/kg [i.e. increase in mean relative liver weight of 39% compared to historical control mean, combined with body weight loss in one female, hunched posture and piloerection in all females (clinical observations) and irregular surface of the forestomach in all females (macroscopic examinations)].
- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

Positive control:

Not examined

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: weighed on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on postnatal day (PND) 1, 4, 7, and 13. Terminal body weights were recorded on the day of scheduled necropsy (fasted for males and non-fasted for females).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not feeding study. Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not drinking water study. Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.

Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for female no. 66 (150 mg/kg Group) that had to be euthanized in extremis and female no. 63 (150 mg/kg Group) with a total litter loss.

Sperm parameters (parental animals):

For the testes of all males of Control Group and 500 mg/kg Group, and all males that failed to sire or died before mating, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible). Selective elimination of pups, e.g. based upon body weight or Anogenital distance (AGD), was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was performed.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

Blood of F1-animals (2 pups/litter) was collected on PND 4 and PND 14-16, if possible. Blood samples at a target volume of 1.0 mL (F0-animals), 0.5 mL (pooled PND 4 pups) and 1.0 mL (PND 14-16 pups) were collected into tubes without anticoagulant. Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4). Measurement of total T4 was conducted for F0-males and PND 14-16 pups.

GROSS EXAMINATION OF DEAD PUPS: Pups that died before scheduled termination were examined externally and sexed (both externally and internally). 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.

Postmortem examinations (parental animals):

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

SACRIFICE
Unscheduled deaths (sacrificed in extremis) were further examined by necropsy and specified tissues were retained but not weighed.

Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted on the following days:
-Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
-Females which delivered: PND 14-16.
-Females which failed to deliver (no. 57): Without evidence of mating: 25 days after the last day of the mating period.
-Females with total litter loss (No. 63): Euthanized within 24 hours after the last pup was found dead or missing.
All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0- females were not fasted.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were collected for all animals and samples for selected animals were prepared for microscopic examination and/or weighed.

The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
-Selected animals and unscheduled deaths (sacrificed in extremis): bone marrow, bone (femur, sternum), brain (eight levels), cervix, epididymides, eye, adrenal gland, coagulation gland, mammary gland, parathyroid, pituitary gland, prostate gland, seminal vesicle, thyroid, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, cecum, colon, rectum, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, sciatic nerve, ovaries, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testes, thymus, trachea, urinary bladder, uterus, vagina.
-Males that failed to sire, females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
-Females with total litter loss: Mammary gland.
-Remaining animals: Gross lesions/masses

Postmortem examinations (offspring):

SACRIFICE
- Pups that died before scheduled termination were examined externally and sexed (both externally and internally). 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. On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two PND 14-16 pups per litter and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues were prepared for microscopic examination and weighed, respectively.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.

Reproductive indices:

From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care. In addition, the following reproduction parameters were determined: mating and fertility indices, precoital time, number of implantation sites, and maternal care.

Offspring viability indices:

The following developmental parameters were determined: gestation index and duration, parturition, sex ratio, live litter size and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 14-16 pups and macroscopy).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Slight salivation after dosing, considered a physiological response to the irritant test item rather than a sign of systemic toxicity, occurred among animals treated at 150 or 500 mg/kg in a dose-related manner. Rales occurred transiently in several treated males (at all dose levels, without a dose-related trend). This respiratory symptom was attributed to the oral gavage administration of the irritant test item.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable - oral gavage study

Mortality:

mortality observed, treatment-related

Description (incidence):

Two treated animals (a low-dose male and a mid-dose female) were sacrificed prematurely for humane reasons. These premature deaths were regarded to be related to the oral gavage administration of the irritant test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A finding of note was the weight loss of one high-dose male (no. 39). This male lost 11% of its initial weight during treatment weeks 1-3 (no further loss in the next week). This was accompanied by hunched posture and laboured respiration on Days 7-11/10. Macroscopic and microscopic examination revealed no obvious cause of the weight loss (no. 39 showed similar gastric changes as most other high-dose males which grew normally; microscopy was limited to the stomach). The weight loss of no. 39 was considered not to reflect an adverse effect of the test item on body weight (gain) since such weight loss occurred in only a single (surviving) animal.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences between treated rats and controls were limited to a lower mean corpuscular haemoglobin (MCH) value in high-dose (500 mg/kg) males (relative difference: -4%). As main red blood cell parameters (haemoglobin concentration, number of red blood cells) of high-dose males were not affected, the lower MCH was considered not to reflect an adverse effect of the test item on red blood cells.
The abnormal white blood cell values noted in low-dose female no. 59 (higher numbers of neutrophils and total white blood cells, lower number of lymphocytes) were likely to be related to a thymoma (which was unrelated to treatment).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes in clinical pathology parameters consisted of increases in cholesterol and bile acids and a decrease in total protein in males treated at 500 mg/kg. In the absence of associated adverse anatomic pathology alterations, these changes were regarded as non-adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The microscopic correlates were inflammation and erosions in the forestomach and glandular stomach, hyperkeratosis (and a few males with squamous cell hyperplasia) in the forestomach, and submucosal edema and hemorrhages in the glandular stomach.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Mean serum level of T4 in males treated at 500 mg/kg was 16% lower than the concurrent control mean. However, as statistical significance was not achieved and mean T4 value in 500 mg/kg treated males remained within the historical control range, this change was attributed to biological variation.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

At 50 mg/kg, mating was not successful for one couple (female no. 57, male no. 16). Prior to the pairing with female no. 57, this male mated successfully with female no. 56 and was therefore not considered to be the cause of the reproduction failure of this pair.
At 150 mg/kg, one female (no. 63) had total litter loss on PND 3. At microscopic examination, slight, focal necrosis was observed in the myometrium which is most likely normal short after delivery. The other reproductive organs showed no abnormalities.
Another female at 150 mg/kg (no. 66, mated with male no. 26) was sacrificed for humane reasons on the day she showed evidence of mating. Her pregnancy status could not be determined due to the brief period between mating and sacrifice. Microscopic examination showed massive hyperkeratosis of her vagina. Her uterus and cervix could not be examined due to severe autolysis (Salewski staining).
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for test item-related abnormal spermatogenesis.

Details on results (P0)

Mating index was not affected by treatment. Precoital time was considered not to be affected by treatment. Number of implantation sites was not affected by treatment. Fertility index was not affected by treatment.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

other: clinical pathology - non-adverse

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 100, 99, 98 and 100% for the control, 50, 150 and 500 mg/kg groups, respectively. A few pups went missing (presumed cannibalized): one at 50 mg/kg (on PND 2, dam no. 52) and two at 150 mg/kg (one of dam no. 63 on PND 3 (total litter loss) and one of dam no. 65 on PND 4). This post-natal loss was regarded as unrelated to treatment due to the incidental occurrence (within the range considered normal for pups of this age) and lack of a dose-related trend.

Body weight and weight changes:

no effects observed

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:

effects observed, non-treatment-related

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.
The statistically significant difference noted in males (higher mean T4 at 50 mg/kg) was regarded as unrelated to treatment due to the lack of a dose-related response.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

-Gestation Index and Duration:
Gestation index and duration of gestation were not affected by treatment. All pregnant females had live offspring (gestation index 100% for all groups).
-Parturition/Maternal Care:
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.
-Post-Implantation Survival Index:
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was considered not to be affected by treatment. The survival indices were 95, 95, 92 and 87% for the control, 50, 150 and 500 mg/kg groups, respectively.
The slightly low post-implantation survival index at 500 mg/kg could be explained by the high number of unaccounted for sites in female no. 78 (6/13 implantation sites were unaccounted for). Moreover, post-implantation survival at 500 mg/kg remained within the historical control range. Therefore, the slightly low post-implantation survival index at 500 mg/kg was regarded as unrelated to treatment.
For control female no. 48 the number of pups born was higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.
-Litter Size:
The number of living pups at first litter check (live litter size) was not affected by treatment. Mean live litter sizes were 11.4, 12.9, 10.4 and 10.6 for the control, 50, 150 and 500 mg/kg groups, respectively.
-Live Birth Index:
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 99, 99, 96 and 100% for the control, 50, 150 and 500 mg/kg groups, respectively.
At first litter check, one control pup (litter no. 45), one 50 mg/kg pup (litter no. 56) and four 150 mg/kg pups (one of litter nos. 63 and 69, two of litter no. 64) were found dead. This pup mortality was considered unrelated to treatment as the incidence showed no dose-related trend and remained within normal limits.
-Lactation Index:
Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect levels (NOAELs) of the test substance were established:
Reproduction/developmental NOAEL: at least 500 mg/kg (highest dose tested).

Executive summary:

In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, Wistar Han rats were treated with undiluted test substance by daily oral gavage at dose levels of 50, 150 and 500 mg solid matter/kg (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received water (Elix). Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (mostly for 50-55 days). A female without offspring (non-mated) was treated for 53 days and a female which had a total litter loss was treated for 42 days.

No reproduction/developmental toxicity was observed up to the highest dose level tested (500 mg/kg). No treatment-related changes were noted in the reproductive parameters examined (i.e., mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Based on the results, the following No Observed Adverse Effect levels (NOAELs) of the test substance were established:

Reproduction/developmental NOAEL: at least 500 mg/kg (highest dose tested).