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

Endpoint summary

Description of key information

The key study for repeated dose 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 general toxicity (Harlan Laboratories 2013).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

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: 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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were analysed 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)

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily.

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10/Sex/dose

Control animals:

yes

Details on study design:

No animals:
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)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily throughout acclimatisation, 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 acclimatisation (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.

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, hematocrit, 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 (Table 1)

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

Sacrifice and pathology:

SACRIFICE
Allocation A animals (Tox and 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 2 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 hematoxylin and eosin. Additionally, section of testes and epididymides were also stained in PAS-hematoxylin.

Statistics:

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

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. The clinical signs observed were considered related to a gavage error but were not considered to have toxicological significance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Unscheduled deaths occurred in three test animals from allocation A females and one control female. Deaths considered to be not treatment-related.

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:

effects observed, treatment-related

Description (incidence and severity):

but minor

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight gain of males in group 4 (allocations A and C) was slightly lower as compared to the control group. However, mean body weights of control males and group 4 males were similar and the slight difference was considered to be incidential. Slight differences in mean body weigh gain in between control group and test item-treated females, but these findings were considered incidential as mean body weights were similar.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

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 oestrous 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.

No test item related FOB findings were observed in any of the measured parameters.

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 observed

Critical 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 reproduction/developmental toxicity).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A repeated dose toxicity study, conducted according to OECD TG 422 and in compliance with GLP has been performed 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 in animals selected for toxicity testing and recovery. 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 haematology and urine parameters, or on macroscopic or microscopic necropsy findings.

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 a 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 the recovery period. 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. 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, 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 (see Section 7.8.1), the difference in uterine weights between control and high dose after 4 weeks was regarded as not adverse.

Justification for classification or non-classification

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