2,4-Hexadienoic acid, 3-(trimethoxysilyl)propyl ester, (2E,4E)-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The key study for reproductive toxicity, conducted according to OECD TG 422 and in compliance with GLP reports a NOAEL of 1000 mg/kg/day (the highest dose level tested) for toxicity to reproduction (Harlan Laboratories 2013).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30.01.2013 - 26.06.2013

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)

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economic Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of Environment (MOE) Guidelines of 31 March 2011.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: (P) 10 weeks
- Weight at study initiation: (P) Males: 305 - 353 g; Females: 185 - 247 g
- Fasting period before study:
- Housing: individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 51
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

62 males and 102 females were obtained from the breeder. The surplus (reserve) animals were sacrificed after the commencement of treatment. This was documented in the raw data.

Group 1: 20 males and 30 females
Group 2: 10 males and 20 females
Group 3: 10 males and 20 females
Group 4: 20 males and 30 females

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

dried and deacidified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Dose concentrations:
Group 1: 0 mg/mL
Group 2: 6 mg/mL
Group 3: 60 mg/mL
Group 4: 200 mg/mL

Dose volume: 5 mL/kg body weight

VEHICLE
- Justification for use and choice of vehicle: the test substance was stable in dried and deacidified corn oil
- Lot/batch no. (if required): 492194511

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of copulation was confirmed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were analyzed using a GC/FID method provided by the Sponsor. Concentration, homogneity and stability (after 8 days) of dose formulations were determined in samples (ca. 1g sample volume) taken after experimental start. Concentration of the dose formulations were also determined in samples of the formulations prepared during the end of the treatment period.

The following acceptance criteria were applied to the analytical results:
1. Concentration: The achieved content should be +/-10% of the nominal content
2. Homogeneity: The individual recoveries should not deviate more than +/-15% from the corresponding mean
3. Stability: The results obtained from storage stability samples should not deviate more than 10% from time-zero reference (content or mean of homogeneity samples)

Duration of treatment / exposure:

28 days (Allocation A males)
29 days (Allocation B and allocation C animals)
ca. 6 weeks (Allocation A females)

Frequency of treatment:

Once, daily.

Details on study schedule:

FEMALES:

Treatment start: Day 1 of pre-pairing = day 1 of treatment
Pre-pairing: 14 days
Pairing: 1-4 days
Gestation: ca. 21 days
Treatment ends: On day 4 post partum; females not giving birth on day 24 post coitum
Blood and urine sampling: None
Necropsy: Dams on day 5 post partum, pups on day 4 post partum; Females not giving birth on day 25 post coitum


MALES:

Treatment start: Day 1 of pre-pairing (day 1 of treatment)
Pre-pairing: 14 days
Pairing: 1-4 days
Treatment ends: On the day before sacrifice
Blood and urine sampling: On the day of necropsy
Necropsy: After treatment for 28 days

Remarks:

Doses / Concentrations:
30 mg/kg/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

10/Sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

GROUP 1 (control)
Males
A: 1 - 10
C: 11 - 20
Females
A: 61 - 70
B: 71 - 80
C: 81 - 90

GROUP 2 (30 mg/kg bw/day)
Males
A: 21 - 30
Females
A: 91 - 100
B: 101 - 110

GROUP 3 (300 mg/kg bw/day)
Males
A: 31 - 40
Females
A: 111 - 120
B: 121 - 130

GROUP 4 (1000 mg/kg bw/day)
A: 41 - 50
C: 51 - 60
Females
A: 131 - 140
B: 141 - 150
C: 151 - 160

A= Animals that were paired for mating; the males were used for both reproduction and toxicity testing (treatment for 28 days); the females were used for reproduction testing
B= Satellite females used for toxicity testing (no mating; treatment for 29 days)
C= Recovery animals (no mating; treatment for 29 days followed by a 14 days recovery period)

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily throughout acclimatization, treatment and recovery period. Viability and mortality were checked twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour were checked weekly in all animals. In Allocation A females the parameters were checked once prior to treatment start, weekly during pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period and on day 3 post partum.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded during acclimatization, on day 1 of the treatment phase and then daily until the end of treatment. During recovery, body weights were recorded on day 1, weekly thereafter and at termination.

OTHER:
FOB testing was conducted during acclimatization (Allocation A males, B females and C males and females), last treatment week (allocation A males and B females) and recovery week 2 (allocation C males and females).This included cage side observations, hand held observations, open field observations, reflexes, and measurements of counts (hind-limb/fore-limb grip strength, body temperature and landing footsplay and locomotor activity.

FOOD CONSUMPTION:
- Food consumption was recorded weekly during treatment and recovery periods for A males, B females and C males and females. No food consumption was recorded for A males during the pairing period. Food consumption for allocation A females was recorded weekly during pre-pairing, gestation days 0-7, 7-10, 10-14, 14-18, 18-21, and days 1-4 post partum. No food consumption was recorded during pairing period.

Blood and urine samples for clinical laboratory investigations were collected after treatment for 28 days for allocation A males, treatment for 29 days for allocation B females, treatment for 29 days followed by 2 weeks of recovery period for allocation C males and females. Blood samples were collected following fasting period of 18 hours and under light isoflurane anesthesia. Urine was collected during the 18-hour fasting period into a specimen vial, using a metabolism cage.
Haematology: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, total leukocyte count, differential leukocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells), platelet count, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, reticulocyte count, reticulocyte maturity index (low, medium, high fluorescence). Methemoglobin, Heinz bodies, coagulation parameters.

Clinical biochemistry parameters: see parameters examined in table 1
Urinanalysis: see parameters examined in table 1

Oestrous cyclicity (parental animals):

Estrous cycle was determined by histopathological examination.

Sperm parameters (parental animals):

Qualitative assessment of the male reproductive organs was performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Postmortem examinations (parental animals):

SACRIFICE
Allocation A animals (Tox/repro males, repro females): Males were sacrificed after treatment for 28 days. The dams were sacrificed on day 5 post partum. Females not giving birth were sacrificed on day 25 post coitum.

Allocation B animals (Tox females): All allocation B animals (satellite females for toxicity testing) were sacrificed after treatment for 29 days.

Allocation C (Recovery) animals: Males and females were sacrificed 2 weeks after the last day of test item administration.

GROSS NECROPSY
All animals were examined macroscopically for any structural changes. Blood samples were taken from all adult animals except reproductive females (Group A), for possible thyroid hormone analysis. Special attention was directed at the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively. Relative organ weights were calculated on the basis of body weight and brain weight. All organ and tissue samples which were collected (Table 1) were processed, embedded, cut and stained with haematoxylin and eosin. Additionally, section of testes and epididymides were also stained in PAS-haematoxylin.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on Day 4 post partum

GROSS NECROPSY
All animals were examined macroscopically for any structural changes.

Statistics:

Mean and median values and SD of various data included in the report, The Dunnett test, The Steel test, Fisher's exact test.

Reproductive indices:

Fertility indices, mean precoital time, post-implantation losses, mean litter size

Offspring viability indices:

Pup sex ratios, viability indices.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation and bedding in mouth of group 4 males after treatment. Considered to be test item-related but not toxicologically significant. One female from group 4 and another one from group 3 showed decreased activity, prostate position, ruffled fur, scabs at both eyes and tachypnea. These effects were considered related to gavage errors and were not considered to have toxicological significance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three unscheduled deaths occurred, one control animal and two test-substance treated animals. The mortalities observed in animals dosed with 300 and 1000 mg/kg bw/day were considered related to gavage errors and were not considered to have toxicological significance.

Body weight and weight changes:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The observed effects were not toxicologically significant.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no test material related mortalities. No test item related clinical signs of toxicological significance were observed at the cage side or detailed examinations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights and body weight gain were considered to be unaffected by treatment with the test item. There were no test item related effects on food consumption.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No test item related effects were observed in estrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test item related effects were observed in mating performance, fertility, corpora lutea count and implantation rate, post-implantation loss and duration of gestation. The gestation indices (number of females with living pups as a percentage of females pregnant) were 90%, 87.5%, 88.9%, 87.5%, respectively at 0, 30, 300 and 1000 mg/kg/day). Litter sizes (at first litter check) and post-natal loss (days 0-4 post partum) were similar in control and test item - treated animals

ORGAN WEIGHTS (PARENTAL ANIMALS)
Increased mean absolute and relative kidney weights were observed in males and females treated at all doses, and decreased absolute uterine weights were observed at 1000 mg/kg/day. The changes were not considered adverse, since there were no corresponding microscopic findings and they were reversible.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test item related macroscopic or microscopic findings.

OTHER FINDINGS (PARENTAL ANIMALS)
No test item related abnormalities were observed during treatment week 4 and during recovery. In addition, there were no test item-related effects on fore- and hindlimb grip strength, body temperature, landing foot splay or locomotor activity.

There was no test item related effect on food consumption.

There were no test item related changes in haematology and urine parameters. The following, test item related changes in clinical chemistry parameters were reported: decreased creatinine levels in males and females at all dose levels, increased cholesterol and phospholipid levels in females with 1000 mg/kg/day, increased plasma sodium, potassium, chloride, calcium and phosphorus in males treated with 300 and/or 1000 mg/kg/day and increased calcium levels in females treated with 30, 300 and 1000 mg/kg/day. Increased total protein and albumin concentrations in males treated with 300 or 1000 mg/kg/day. The sodium level was still increased in males after recovery. All test item related changes in clinical chemistry were considered minor and non-adverse.

MATING PERFORMANCE AND FERTILITY
No test item-related effects on mating performance and fertility were observed. All females were mated during the first pairing period. All females in the control group were pregnant. No test item-related effects on the number of corpora lutea were observed. The mean duration of gestation was unaffected by exposure to the test item. There were no test item related effects on implantation rate or post implantation loss. There were no test item related effects on litter size. There was no test-item related effect on postnatal loss.
Based on the results of this study, 1000 mg/kg/day (the highest dose level tested) is the NOAEL (no-observed-adverse-effect-level) for general toxicity and the NOEL (no-observed-effect level) for reproduction/developmental toxicity).

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 were attributed to test material administration.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
No test-item related findings were noted for pups during first litter check and lactation. Sex ratios of pups were unaffected by exposure to the test item.

CLINICAL SIGNS (OFFSPRING)
No test-item related findings were noted for pups during first litter checks and lactation.

BODY WEIGHT (OFFSPRING)
There were no test item related effects on pup body weights and body weight gain.

GROSS PATHOLOGY (OFFSPRING)
No test item-related abnormalities were observed at necropsy of pups.


OTHER FINDINGS (OFFSPRING)
Sex ratio of pups was unaffected by exposure to the test item.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test material related effects were observed.

Reproductive effects observed:

no

Conclusions:

Based on the results of this study, 1000 mg/kg/day (the highest dose level tested) is the NOAEL (no-observed-adverse-effect-level) for general toxicity and the NOEL (no-observed-effect level) for reproductio or developmental toxicity).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A reproductive screening study, conducted according to OECD TG 422 and in compliance with GLP has been conducted via the oral route with the submission substance (Harlan Laboratories 2013). The test item was administered in dried and deacidified corn oil at 0, 30, 300 and 1000 mg/kg bw/day. The duration of treatment was 4 weeks with 2 weeks recovery period in animals selected for toxicity testing and recovery. Females used for testing of reproduction were treated for ca. 6 weeks (14 days prior to pairing, through pairing, gestation ad lactation until F1 generation reached day 4 post partum). Pups were sacrificed on day 4 post partum. Treatment with test item had no effect on mortality, food consumption or body weight. No test item related clinical signs of toxicological significance and no test item related effects on functional observation battery parameters were reported. Similarly, there were no test item related effects in hematology and urine parameters, or on macroscopic or microscopic findings.

 

The treatment with test item did not affect reproduction and development. Some test item-related changes in clinical chemistry parameters were observed after 4 weeks of treatment, including electrolyte (sodium, chloride, calcium, potassium, phosphorus) plasma concentrations and increased protein and albumin levels in males at 300 and/or 1000 mg/kg/day, increased calcium levels in females at ≥30 mg/kg/day and increased cholesterol and phospholipid levels in females at 1000 mg/kg/day. The changes were minor and within historical value ranges and similar to controls, most of having reversed during 14 day treatment free recovery period. Only the sodium concentration of males treated at 1000 mg/kg/day was still increased as compared to the control group after recovery. The changes in plasma chemistry parameters were considered to be not adverse.

 

After 4 weeks of treatment, absolute and/or relative kidney weights were increased in males and females at ≥30 mg/kg/day (only in animals used for toxicity testing). Since there were no corresponding microscopic findings, there were no test item related effects on standard urine parameters and no similar observation was made in recovery animals, the slightly increased kidney weights in test item treated animals were considered to be not adverse.

 

In females treated with 1000 mg/kg/day (toxicity testing), decreased absolute and relative uterine weights were recorded after 4 weeks. No corresponding histological alterations were observed and no similar observation was made in recovery animals. Considering that no test item related effect on reproduction was observed, the difference in uterine weights between control and high dose after 4 weeks was regarded as not adverse.

Effects on developmental toxicity

Description of key information

The key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, conducted according to OECD 422, and in compliance with GLP, reports a NOAEL of 1000 mg/kg/day (the highest dose level tested) for developmental toxicity (Harlan Laboratories, 2013).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-30 to 2013-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

yes

Remarks:

The deviation according to the latest amendmend of the guideline is that the maternal animals were sacrificed on day 4 post partum.

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economic Trade and Industry (METI), Ministry of Health, Labour and Welfa re (MHLW) and Ministry of Environment (MOE) Guidelines of 31 March 2011

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 10 weeks
- Weight at study initiation: 305-353 g (males) and 185-247 g (females)
- Fasting period before study:
- Housing: individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding
- Diet: Pelleted standard rodent maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 51
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

62 males and 102 females were obtained from the breeder. The surplus (reserve) animals were sacrificed after the commencement of treatment. This was documented in the raw data.
Group 1: 20 males and 30 females
Group 2: 10 males and 20 females
Group 3: 10 males and 20 females
Group 4: 20 males and 30 females

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Dried and deacidified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose concentrations:
Group 1: 0 mg/mL
Group 2: 6 mg/mL
Group 3: 60 mg/mL
Group 4: 200 mg/mL

Dose volume: 5 mL/kg body weight

VEHICLE
- Justification for use and choice of vehicle: The test substance was stable and soluble in dried and deacidified corn oil
- Lot/batch no. (if required): 492194511

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were analyzed using a GC/FID method provided by the Sponsor. Concentration, homogeneity and stability (after 8 days) of dose formulations were determined in samples (ca.1g sample volume) taken after experimental start. Concentration of the dose formulations were also determined in samples of the formulations prepared during the end of the treatment period.
The following acceptance criteria were applied to the analytical results:
1. Concentration: The achieved content should be +/-10% of the nominal content
2. Homogeneity: The individual recoveries should not deviate more than +/-15% from the corresponding
mean
3. Stability: The results obtained from storage stability samples should not deviate more than 10% from
time-zero reference (content or mean of homogeneity samples)

Details on mating procedure:

- Impregnation procedure:cohoused
- If cohoused: females were housed with sexually mature males until evidence of copulation was observed
- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of copulation was confirmed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually

Duration of treatment / exposure:

28 days (Allocation A males)
29 days (Allocation B and allocation C animals)
ca. 6 weeks (Allocation A females)

Frequency of treatment:

Once, daily

Duration of test:

Until 4 days post partum

Dose / conc.:

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

Remarks:

Group 1

Dose / conc.:

30 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:

Group 1: 20 males and 30 females
Group 2: 10 males and 20 females
Group 3: 10 males and 20 females
Group 4: 20 males and 30 females

GROUP 1 (control)
Males
A: 1 - 10
C: 11 - 20
Females
A: 61 - 70
B: 71 - 80
C: 81 - 90
GROUP 2 (30 mg/kg bw/day)
Males
A: 21 - 30
Females
A: 91 - 100
B: 101 - 110
GROUP 3 (300 mg/kg bw/day)
Males
A: 31 - 40
Females
A: 111 - 120
B: 121 - 130
GROUP 4 (1000 mg/kg bw/day)
A: 41 - 50
C: 51 - 60
Females
A: 131 - 140
B: 141 - 150
C: 151 - 160
A= Animals that were paired for mating; the males were used for both reproduction and toxicity testing (treatment for 28 days); the females were used for reproduction testing
B= Satellite females used for toxicity testing (no mating; treatment for 29 days)
C= Recovery animals (no mating; treatment for 29 days followed by a 14 days recover)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous non-GLP 14-day dose range-finding toxicity study in Sprague Dawley rats, using dose levels of 150, 400 and 1000 mg/kg bw/day. No adverse test item-related effects were observed.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during the acclimatization period, treatment (within 3 hours post-dose) and recovery periods
- Cage side observations checked included: viability, mortality and behavioral abnormalities changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity, changes in gait, posture and response to handing as well as the presence of clonic and tonic movements, stereotypes or bizarre behavior.

DETAILED CLINICAL OBSERVATIONS: Yes; changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity, changes in gait, posture and response to handing as well as the presence of clonic and tonic movements, stereotypes or bizarre behavior.
- Time schedule: weekly starting from the treatment period on all allocation A males, allocation B females and recovery (allocation C) animals. Detailed clinical observations were also performed on allocation A females once prior to treatment start, weekly during the pre-paiting and pairing periods, on days 0, 6, 13 and 20 of the gestation period, and on day 3 post partum.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded during acclimatization, on day 1 of the ttreatment phase and then daily until the end of treatment. During recovery body weights were recorded on day 1, weekly thereafter and at termination

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 5 post partum; females not giving birth were sacrificed on day 25 post coitum
- Organs examined: see table 1

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes, all pups
- Gross anomalies: Yes, all pups
- The sex ratio of the pups was recorded
- Pups were weighed individually on days 0, 1 and 4 post partum

Statistics:

Mean and median values and SD of various data included in the report, The Dunnett test, The Steel test, Fisher's exact test.

Indices:

Fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios, viability indices

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs.
Decreased activity, prostrate position, ruffled fur, scabs at both eyes and tachypnea were observed prior to death in two reproduction test females that were sacrificed prior to scheduled death. These clinical signs observed were considered to be related to a gavage error. No other clinical signs were noted in any of the test animals.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two reproduction testing females from allocation group A and one control female died during the study, prior to scheduled death. Two of the animals allocation group A animalsl) exhibited moribund condition prior to death, which was concluded to be related to the gavage treatment. The death of the control animal could not be established.

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:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changed in haematology parameters.
Methemoglobin levels were increase in males and females treated with 30 or 300 mg/kg bw/day and in females treated with 1000 mg/kg bw/day after 4 weeks of treatment. After recovery, methemoblobin levels of females treated with 1000 mg/kg bw/day were slightly lower than in the control group. Slight decrease in mean relative number of large unstained cells in females of 1000 mg/kg bw/day group after recovery. These differences were considered to be incidential since they were slight and contradictory in males and females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in creatinine, cholesterol, phospholipids and calcium levels were observed in group 4 (1000 mg/kg bw/day) females after 4 weeks of treatment. All test item-related changes were considered minor. The values were within the range of the historical control data and/or they differed slightly from values obtained from control animals after the treatment or recovery period.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

In group 2 females (30 mg/kg bw/day) the protein concentration was significally lower to the control group values after the treatment. This effect was considered to be incidential as there was no dose-response relationship.

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):

Increased mean absolute and relative kidney weights were observed in males and females treated at all doses, and decreased absolute uterine weights were observed at 1000 mg/kg/day. The changes were not considered adverse, since there were no corresponding microscopic findings and they were reversible.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item related macroscopic or microscopic findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related findings at histopathology.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

One female treated with 1000 mg/kg bw/day and one female treated with 30 mg/kg bw/day delivered only dead pups.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Dead pups were observed at first litter check: 1/16 pups of a control female, 3/15 pups of a 300 mg/kg bw/day female and 10/10 pups of a 1000 mg/kg bw/day female. This effect was considered to be incidential and not treatment-related.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights of pups in groups 3 and 4 (300 and 1000 mg/kg bw/day) were slightly lower when compared to the control group. However, the differences were not statistically significant and were considered to be incidential and not related to the treatment.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

4 pups from control group, 1 pup from group 2 (30 mg/kg bw/day), 10 pups from group 3 (300 mg/kg bw/day) and 3 pups in group 4 (1000 mg/kg bw/day) died during days 0-4 post partum. These effects were considered to be incidential since there was no dose-response relationship.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Lack of milk in the stomach was observed for pups from groups 1, 3 and 4. No test-item related findings were noted.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not examined

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 observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Based on the results of this study, 1000 mg/kg/day (the highest dose level tested) is the NOAEL (no-observed-adverse-effect-level) for general toxicity and the NOEL (no-observed-effect level) for reproductio or developmental toxicity).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

A reproductive screening study, conducted according to OECD TG 422 and in compliance with GLP has been conducted via the oral route with the submission substance (Harlan Laboratories 2013). The test item was administered in dried and deacidified corn oil at 0, 30, 300 and 1000 mg/kg bw/day. The duration of treatment was 4 weeks and 2 weeks recovery period in animals selected for toxicity testing and recovery. Females used for testing of reproduction were treated for ca. 6 weeks (14 days prior to pairing, through pairing, gestation ad lactation until F1 generation reached day 4 post partum). Pups were sacrificed on day 4 post partum. Treatment with test item had no effect on mortality, food consumption or body weight. No test item related clinical signs of toxicological significance and no test item related effects on functional observation battery parameters were reported. Similarly, there were no test item related effects in hematology and urine parameters, or on macroscopic or microscopic findings.

 

The treatment with test item did not affect reproduction and development. Some test item-related changes in clinical chemistry parameters were observed after 4 weeks of treatment, including electrolyte (sodium, chloride, calcium, potassium, phosphorus) plasma concentrations and increased protein and albumin levels in males at 300 and/or 1000 mg/kg/day, increased calcium levels in females at ≥30 mg/kg/day and increased cholesterol and phospholipid levels in females at 1000 mg/kg/day. The changes were minor and within historical value ranges and similar to controls, most of having reversed during 14 day treatment free recovery period. Only the sodium concentration of males treated at 1000 mg/kg/day was still increased as compared to the control group after recovery. The changes in plasma chemistry parameters were considered to be not adverse.

 

After 4 weeks of treatment, absolute and/or relative kidney weights were increased in males and females at ≥30 mg/kg/day (only in animals used for toxicity testing). Since there were no corresponding microscopic findings, there were no test item related effects on standard urine parameters and no similar observation was made in recovery animals, the slightly increased kidney weights in test item treated animals were considered to be not adverse.

No treatment-related effects were observed in pups during the 4 -day observation period. Females from the control group, group 3 (300 mg/kg bw/day) and group 4 (1000 mg/kg bw/day) delived dead pups. 4 pups from control group, 1 pup from group 2 (30 mg/kg bw/day), 10 pups from group 3 (300 mg/kg bw/day) and 3 pups from group 4 (1000 mg/kg bw/day) died during days 0-4 post partum. Mean body weights of pups in groups 3 and 4 (300 and 1000 mg/kg bw/day) were slightly lower when compared to the control group. Lack of milk in the stomach of pups from groups 1, 3 and 4 was observed during macroscopic examinations. No abnormalities were observed at histopathology. The reported effects were considered to be incidential since no dose-response relationship was noted.

Justification for classification or non-classification

Based on the available information, no classification is required for reproductive or developmental toxicity in accordance with Regulation (EC) No. 1272/2008.