3,5,5-trimethylhexyl acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on a valid OECD 414 compliant study an oral NOAEL of 40 mg/kg bw/day was determined.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2012-05-30 to 2013-04-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories BV, Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: 314 to 361 g (males), 181 to 208 g (females)
- Fasting period before study: no
- Housing: in groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: at room temperature (20±5 °C) in glass beakers

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 0, 10, 31.25, 100 mg/mL
- Amount of vehicle: 4 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days maximum
- 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 (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (7 days) samples of about 2 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. During the last week of the treatment, samples were taken from the middle to confirm concentration.
The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to analytic laboratory and stored there at -20 ± 5 °C until analysis.
The samples received were dissolved in TBME by sonication for at least 5 minutes and then diluted to volume with TBME. Sample solutions were further diluted with TBME into the calibration range. The samples were analysed with gas chromatography.

Duration of treatment / exposure:

Neononyl Acetate was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Frequency of treatment:

daily

Details on study schedule:

not relevant

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats using dose levels of 0, 100, 300 and 1000 mg/kg bw/day, resulting in death at 1000 mg/kg bw/day as well as decreased body weight gain at 300 mg/kg bw/day.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was prepared once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

BODY WEIGHT: Yes
- Time schedule for examinations: daily from treatment start to day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Males: Pre-pairing period, days 1-8 and 8-13, and days 1-3 after pairing period
- Females: Pre-pairing period, days 1-8 and 8-13; gestation period, days 0-7, 7-14 and 14-21, and days 1-4 of the lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OTHER: HEMATOLOGY, CLINICAL CHEMISTRY, NEUROBEHAVIOURAL EXAMINATION

Oestrous cyclicity (parental animals):

Not examinated

Sperm parameters (parental animals):

Not examinated

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed after treatment for 28 days
- Maternal animals: All surviving animals were sacrificed on day 5 post partum

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

HISTOPATHOLOGY / ORGAN WEIGHTS
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately. In addition, from 5 males and females killed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)
- Epididymides (in Bouin’s fixative)
The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries

In addition, from the five males and females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Gross lesions
- Brain
- Spinal chord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to post mortem examinations (macroscopic and/or microscopic examination) as follows:
For the pups either at planed termination or during lactation if dead occurred, special attention was directed at heart, coronary blood vessels and large blood vessels near the heart.

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

Statistics:

The following statistical methods were used to analyze food consumption, body weights, locomotor activity, grip strength, body temperature, clinical laboratory investigations, reproduction data, organ weights and ratios as well as macroscopical findings:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated: mean precoital time, percentage mating, fertility index, conception rate, post-implantation loss, gestation index, birth index and viability index.

Offspring viability indices:

From the on-line reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratio and viability index.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All males survived until the scheduled necropsy.
At the dose level of 400 mg/kg bw/day, one female (no. 84) showed continuous body weight loss from the treatment start and had weakened condition together with severely decreased activity and ruffled fur on day 13 of the pre-pairing period. These findings triggered early termination of the female for humane reasons on day 13 of the pre-pairing period. Based on findings during histopathological examination (ulceration of the forestomach) a perforation during gavage was considered to be possible reason for the moribund condition of this female.
Eight further females at the high-dose level were found dead during gestation period: female nos. 78, 79, 81, 82, 83, 86, 87 and 88 on days 21, 18, 20, 19, 20, 21, 21 and 23 p.c., respectively. Female no. 79 had ruffled fur and decreased activity on the day before its death. Female no. 88 had decreased activity, ruffled fur and chromodacryorrhea for one or two days before death. No severe observations which would indicate bad condition were noted in the remaining females.
The deaths occurred at the end of the gestation period or shortly after the day of the expected birth. All these females were pregnant and embryos or fetuses were found in their uteri during the necropsy. For these reason, difficult parturition was considered to be reason for the early deaths. This assumption was supported by the observation that two females at the high dose level which were not pregnant, survived scheduled study period without any adverse effects.
At the dose level of 125 mg/kg bw/day, two females (nos. 69 and 71) were found dead on days 22 and 23 of the gestation period, respectively. Both females were pregnant and died around the days of the expected birth. Fetuses were found in uteri of these females during the necropsy. Similar to the assumption concerning deaths at the high dose levels, also at the mid-dose level birth complications were considered to be the cause of death.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

All males survived until the scheduled necropsy. One female at high dose was terminated for humane reasons and 8 females were found dead during gestation period. At the mid dose level, two females were found dead during gestation period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males
Treatment with the test item at the dose level of 400 mg/kg bw/day caused body weight loss by 4.7% until the day 4 of the pre-paring period. Statistically significantly reduced body weight gain was noted from day 2 to 14 of the pre-pairing period. Afterwards, body weight gain recovered and was statistically significantly higher than the respective control values from day 6 to 12 of the pairing period and on day 3 of the after pairing period. Absolute body weights were reduced starting from day 3 of the pre-pairing period until the completion of the study. This reduction was statistically significant from day 3 to 12 of the pre-pairing period and on days 1, 2 and 4 of the pairing period. Afterwards, absolute body weights recovered and, although remained slightly lower than the respective control values, the differences were not longer statistically significant. At the dose level of 125 mg/kg bw/day, body weight gain was slightly reduced during the pre-pairing period; the reduction was statistically significant on day 12. Absolute body weights were similar to the control values during the entire study period. At the dose level of 40 mg/kg bw/day, body weight gain and absolute body weights were not affected by the treatment with the test item.
Mean differences in body weight gain at the dose levels of 0, 40, 125 and 400 mg/kg bw/day were +14.3%, +13.6%, +11.2% and +7.2% during the pre-pairing period, +7.7%, +9.5%, +7.9% and +11.2% during the pairing period and +0.8%, +1.8%, +1.5% and +2.2% during the after pairing period (percentages refer to the body weight gain within the respective period).
Treatment with the test item at the dose level of 400 mg/kg bw/day caused statistically significant reduction in food consumption from day 1 to 8 of the pre-pairing period. Mean food consumption during this period was 19.6 g/animal/day at the high dose level compared to 23.5 g/animal/day in the control group. Afterwards food consumption in males at the high dose level recovered and was similar to the respective control value (23.7 g/animal/day compared to 23.4 g/animal/day in the control group) from day 8 to 13 of the pre-pairing period and statistically significantly higher then the respective control value (23.3 g/animal/day compared to 19.3 g/animal/day in the control group) from day 1 to 3 of the after pairing period.
Food consumption in males at the dose levels of 125 and 40 mg/kg bw/day was similar to the respective control values during the entire study period.
Mean differences in food consumption at the dose levels 40, 125 and 400 mg/kg bw/day were respectively: +1.3%, -1.3% and -8.1% during the pre-pairing period and +4.7%, -1.0% and +20.7% during the after pairing period (percentages refer to the respective values in the control group).

Females
Treatment with the test item at the dose level of 400 mg/kg bw/day caused body weight loss by 5.3% until the day 4 of the pre-paring period. In general, body weight gain was statistically significantly reduced from day 2 to 11 of the pre-pairing period. During the remaining pre-pairing period body weight gain remained lower if compared to the control values but the differences were not statistically significant. Body weight gain recovered and was slightly higher than the control values during the gestation period up to day 16 but without statistical significance. Absolute body weights were statistically significantly reduced from day 2 to 11 of the pre-pairing period. Afterwards body weights of females remained lower if compared to the control values until the termination of the group but the differences were not statistically significant. At the dose level of 125 mg/kg bw/day, body weight gain and absolute body weights were periodically slightly lower than the respective control values but the differences were not statistically significant at any time. At the dose level of 40 mg/kg bw/day, body weight gain and absolute body weights were not affected by the treatment with the test item.
Mean differences in body weight gain at the dose levels of 0, 40, 125 and 400 mg/kg bw/day were +7.2%, +9.7%, +8.3% and +3.2% during the pre-pairing period and +49.5%, +52.6%, +44.6% and +36.7% during the gestation period. During the lactation period, mean differences in body weight gain at the dose levels of 0, 40 and 125 mg/kg bw/day were +2.6%, +5.4% and +0.6% (percentages refer to the body weight gain within the respective period).
Treatment with the test item at the dose level of 400 mg/kg bw/day caused statistically significant reduction in food consumption from day 1 to 8 of the pre-pairing period. Mean food consumption was 11.2 g/animal/day at the high dose level compared to 15.7 g/animal/day in the control group during this period. Afterwards food consumption in females recovered and was similar to the respective control value; it was 15.6 g/animal/day compared to 15.0 g/animal/day in the control group from day 8 to 13 of the pre-pairing period. Initially, during the gestation period food consumption in females at the high dose level was higher than the control values, this difference was statistically significant from day 7 to 14 of the gestation period. Mean food consumption was 20.0 g/animal/day at the high dose level compared to 18.4 g/animal/day in the control group. Value of mean food consumption from day 14 to 21 of the gestation period was calculated only for four females because of mortality at the high dose level. Mean food consumption was 17.5 g/animal/day at the high dose level and 19.6 g/animal/day in the control group during this period, the change was statistically significant. At the dose level of 125 mg/kg bw/day, food consumption was similar to the control values during pre-pairing and gestation periods. Statistically significant reduction in food consumption was noted during lactation period. Mean food consumption during this period was 18.6 g/animal/day at this dose level compared to 25.8 g/animal/day in the control group. Food consumption in females at the dose level of 40 mg/kg bw/day was similar to the control values during the entire study period.
Mean differences in food consumption at the dose levels 40, 125 and 400 mg/kg bw/day were respectively: -2.0%, -5.9% and -12.4% during the pre-pairing period and -2.2%, -0.5% and +1.6% during the gestation period. During lactation period, mean differences in food consumption at the dose levels 40 and 125 mg/kg bw/day were +3.5% and -27.9% (percentages refer to the respective values in the control group).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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):

Kidneys
Hyaline droplet nephrophathy consisting of increased tubular hyaline droplets, tubular degeneration and various instances of granular casts was recorded in males at the dose levels of 400, 125 and 40 mg/kg bw/day. The nephropathy is deemed to be related to treatment and is considered to represent an adverse change for the rat only. Since the hyaline droplets in the male rat relate to accumulation of alpha2-micoglobulin, and little or none of this protein is present in man, a nephropathy in man that involves the same mechanism is unlikely to occur with the test item and has limited relevance for other species including man.

Thyroid glands
Follicular hypertrophy was recorded in at substantially elevated incidence in males at the dose levels of 400, 125 and 40 mg/kg bw/day and females at the dose level of 125 mg/kg bw/day. It can be assumed that existing hypertrophic changes in females at the dose level of 400 mg/kg bw/day were masked by post mortal tissue changes since most of the animals in this group prematurely died. The finding is deemed to be associated with increased hepatic turnover of thyroid hormones due to hepatocellular hypertrophy and, therefore, deemed to represent a secondary change.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations. Individual lesions recorded were within the range of background alterations that may be recorded in this type of study, in rats of this strain and age.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

At the dose level of 125 mg/kg bw/day, statistically significant increase of total post-implantation loss was noted.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

Key result

Dose descriptor:

NOEL

Remarks:

for reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

reduced viability index at 125 mg/kg bw/day: 89.1 % compared to 98.0 % in the control group.

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Remarks:

for development

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Treatment with the test item at the dose level of 125 mg/kg bw/day caused increased postnatal loss and reduced viability index.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The No Observed Effect Level (NOEL) for development and reproduction is established at the dose level of 40 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was 40 mg/kg bw/day (was also the NOEL).

Executive summary:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Neononyl Acetate to rats. Neononyl Acetate was administered in corn oil as vehicle at dosages of 40, 125 and 400 mg/kg body weight/day, and controls received the vehicle only. Neononyl Acetate was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Mortality

Treatment with the test item caused mortality of females at the dose levels of 400 and 125 mg/kg bw/day. Eight females at the high-dose level and two females at the mid-dose level were found dead between days 18 and 23 post coitum. With exception for two females at the high dose levels, no severe observations which would indicate bad condition were noted in any of these females. Because the deaths occurred shortly before or during the time of expected birth and fetuses were found in the uteri during necropsy, pregnancy complications and/or difficult parturition were considered to be reason for the early deaths. This assumption was supported by the observation that two females at the high dose level which were not pregnant, survived scheduled study period without any indication of adverse toxicity. One further female at the high-dose level was terminated because of moribund condition during the pre-pairing period. Findings during histopathological examination indicated stomach perforation during gavage as a cause of death. Therefore this death was not test item-related.

General Toxicity

At the dose level of 400 mg/kg bw/day, signs of general toxicity were noted in males and females. Ruffled fur was noted in several males and females on individual days during the pre-pairing period. In both genders, reduction in food consumption, body weight gain and body weights were noted. In males, effects on food consumption, body weight gain and absolute body weights were noted predominantly during the pre-pairing period. Afterwards, food consumption and body weight gain recovered and was similar or higher then the respective controls during the remaining study period, absolute body weights remained lower but the differences were not longer statistically significant. In females, food consumption, body weight gain and absolute body weights were reduced predominantly during the pre-pairing period and recovered thereafter. At the end of the gestation period a second decrease in these values was observed. Reversible effects on food consumption, body weight gain and body weights observed during pre-pairing period in females were considered not to be adverse. The second drop of these values at the end of the gestation period was considered to probably be related to difficult parturition. Changes in body weight gain and absolute body weights might also indicate a partial abortion in the females.

Further test item-related effects at the high-dose level were recorded during clinical laboratory investigations: reduction in the number of total leukocytes, lymphocytes and large unstained cells in females, reduction in the amount of bilirubin in both genders and reduction in concentration of triglicerydes in males. During necropsy, increase in liver weights was noted in males as well as macroscopical changes in the liver (tan discoloration, accentuated lobular pattern, thickened organ) in females. These findings were considered to be an adaptive change and therefore not adverse. During histopathological examination, test item-related findings were recorded in the liver, kidneys and thyroid glands of animals at the dose level of 400 mg/kg bw/day. Histopathological changes were considered to be of non-adverse nature. At the dose level of 125 mg/kg bw/day, no adverse signs of general toxicity were noted in males or females. In both genders, food consumption was similar to the control values during the most of the study period. Significant reduction of food consumption was recorded only for females during lactation period. In both genders, body weight gain was slightly lower than the respective controls whereas absolute body weights at this dose level were similar to the respective control values. These effects were considered to be test item-related but not adverse. During clinical laboratory investigations, reduction in total amount of bilirubin was noted in females at the mid-dose level. During necropsy, tan discoloration of the liver found in one females was considered to be an adaptive change and therefore not adverse. At the dose level of 40 mg/kg bw/day, no test item, related effects were noted in males or females. Histopathological changes were considered to be adaptive (liver findings), specific for the rat

and with limited relevance to human (kidney findings) or to be secondary to hepatocellular hypertrophy (thyroid glands findings) and therefore they were considered not to be adverse.

Reproduction and Development

Mating performance, fertility, number of corpora lutea and duration of gestation were considered not to be affected by the treatment with the test item at any dose level. At the dose level of 400 mg/kg bw/day, number of implantation sites calculated for females which died before giving birth was lower than the control value and below the historical control range. Because at the dose level of 400 mg /kg bw/day, all pregnant females died prior to the scheduled necropsy, several reproduction parameters were not evaluated for this dose group. Treatment with the test item at the dose level of 125 mg/kg bw/day caused increase in post implantation loss resulting in reduction of litter size at first litter check and consequent reduction in birth index. Further, increased postnatal loss and reduced viability index were also noted at this dose level. These effects were considered to be test item-related and adverse. At the dose level of 40 mg/kg bw/day, no effects on relevant reproduction parameters were noted.

Conclusion

The NOEL (No Observed Effect Level) for development and reproduction was established at the dose level of 40 mg/kg bw/day. In females test item-related mortality was noted at the dose levels of 400 and 125 mg/kg bw/day and therefore NOAEL (No Observed Adverse Effect Level) as well as NOEL for general and maternal toxicity was considered to be 40 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP according to OECD TG 422.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

- Reproductive toxicity:

In reproduction/developmental screening study with 3,5,5-trimethylhexyl acetate (Dettwiler 2013), LOAEL for reproductive/developmental effects was determined at 125 mg/kg bw/day. Since the LOAEL for general parental toxicity was considered 125 mg/kg bw/day, i.e. the same level as reproductive effects, it could be concluded that 3,5,5-trimethylhexyl acetate does not need to be classified as reproductive toxicant.

- Waiving of extended one-generation reproductive toxicity study:

An extended one-generation reproductive toxicity study having regard to the likely route of human exposure, is to be conducted for substances within the tonnage band 100 - 1000 tpa, if the 28-day or 90-day study indicates adverse effects on reproductive organs or tissues. Data of three valid repeated dose studies are not indicating any treatment-related adverse effects on fertility. Thus, no adverse effects to the reproductive system are expected. Further testing would therefore most likely not lead to other results and hazard assessment of the substance would not be improved. The available data are considered reliable and sufficient and therefore conduction of an extended one-generation study is not required, also taking animal welfare reasons into account.

Effects on developmental toxicity

Description of key information

Based on the results of a OECD 414 compliant study, there was no embryolethality, fetotoxicity, or signs of teratogenicity at doses up to 250 mg/kg bw/day of test item when administered orally via gavage to pregnant Sprague Dawley rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline study

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

Developmental / teratogenicity study:

The objective of the study was to investigate the embryo-fetal developmental toxicity of the test item administered to pregnant female Sprague-Dawley rats by oral (gavage) daily from Gestation Days (GD) 5 to 20 inclusive. The test item and reference item/vehicle were administered daily, during the dosing period, as shown below:

Table 1. Dosing

Treatment Group	Dose Level (mg/kg bw/day)	Dose Conc. (mg/mL)	Dose Volume (mL/kg)	Number of Pregnant Females
1. Control*	0	0	4	24
2. Test item -Low Dose	15	3.75		24
3. Test item -Mid Dose	50	12.5		24
4. Test item -High Dose	250	62.5		24

*Group 1 animals received the reference item/vehicle (corn oil).

 

The test item was formulated as solutions in corn oil (also the control item/vehicle) and administered via oral gavage to pregnant Sprague-Dawley rats that were roughly 9-10 weeks of age, with body weights ranging from 196 to 263 grams at the start of treatment (November 1, 2015). Animals had been mated on the day prior to shipping, and on the following day, those females confirmed as mated (with vaginal plugs) were shipped. The day of receipt (day following mating) was considered Day 0 of the study. Cage side clinical observations were performed twice daily and a detailed clinical observation was performed on days of body weight recordings. Individual body weight was measured on Days 0, 3, 5, 6, 9, 12, 15, 18 and 21 of gestation. Food consumption was measured on Days 0 to 3, 3 to 5, 5 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 21 of gestation. On GD 21, the animals were euthanized by exsanguination following CO2 asphyxiation and an external macroscopic examination, including identification of all clinically-recorded lesions, as well as a detailed internal examination were performed. The reproductive tract was dissected out, the ovaries removed and corpora lutea counted. The gravid uterus was weighed. The number of implantations recorded and the pre- and postimplantations losses calculated. The uterine contents, including the placenta, were examined and the numbers of live fetuses, dead fetuses, early middle and late resorptions were recorded. Test item-related effects were noted in dams at the high dose only and included mortality in 2 of 24 animals with associated clinical signs of reduced activity, cold to touch, lying on cage floor, and decreased respiration rate, reduced food consumption/body weight, and gross changes at necropsy that included liver findings of pale discoloration/pale area, enlargement and/or prominent lobular architecture, as well as small spleen, gelatinous pancreas, and dark foci/discoloration in the stomach. There were test item-related reduced fetal body weights at the high dose; however, no effects on the number of corpora lutea, implantation sites, live fetuses, sex ratio, resorptions, or pre- and post-implantation losses. There were no incidences of major malformations, minor external or internal anomalies that were related to Neononyl Acetate at any dose levels up to 250 mg/kg/day. The incidence of incomplete ossification of the frontal, interparietal, pubis and parietal bones were significantly increased for both litters and fetuses along with a significant increase of fetuses with incomplete ossification of the supraoccipital bones at 250/mg/kg bw/day; these were considered secondary to maternal toxicity. Also noted were increase of fetuses with incomplete ossification of the ilium and femur at 50 mg/kg bw/day that was considered incidental. In summary, the administration of test item by oral gavage daily from Gestation Days 5 to 20 (inclusive) to pregnant Sprague-Dawley rats at doses of 0, 15, 50, and 250 mg/kg bw/day resulted in maternal mortality, and reduced food consumption/body weight, body weight gain, and corrected body weight, along with gross pathological observations primarily in the liver, for dams at 250 mg/kg bw/day. There were also treatment-related reductions in male, female and total fetal weights and minor skeletal anomalies considered secondary to maternal toxicity generally at 250 mg/kg bw/day. Based on the results of this study, there was no embryolethality, fetotoxicity, or signs of teratogenicity at doses up to 250 mg/kg bw/day of test item when administered orally via gavage to pregnant Sprague Dawley rats.

Justification for classification or non-classification

- Reproductive toxicity:

Based on the above stated assessment of the reproduction toxicity potential of 3,5,5-trimethylhexyl acetate is deemed not to be toxic to the reproduction and accordingly does not need to be classified according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.

- Developmental toxicity / teratogenictiy:

Based on the above stated assessment of the developmental toxicity / teratogenicity potential of 3,5,5-trimethylhexyl acetate is deemed not to be toxic to the developmental organism and accordingly does not need to be classified according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.