Benzoic acid, C12-15-alkyl esters

Endpoint summary

Description of key information

Based on the results of the present Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.

 

Oral administration of the structurally related source substance Benzoic Acid Isononylester, to rats, by gavage, for a period of 28 consecutive days resulted in treatment related changes in either sex at a dose level of 1000 mg/kg/day and in males treated with 150 mg/kg/day.

 

In a subchronic repeated dose toxicity study with the source substance Benzoic Acid Isononylester, there was no evidence of treatment related effects in either sex treated with 300 mg/kg/day, which was the highest administered dose level.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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

Study period:

04 February 2021 - 27 May 2021

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)

Version / remarks:

adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 Italia S.p.A., Calco (Lecco), Italy
- Females nulliparous and non-pregnant: yes
- Age at receipt: approximately 7 to 8 weeks
- Weight at receipt: 212 to 229 g (males), 178 to 208 g (females)
- Housing: from arrival to pairing animalswere housed up to 5 of one sex to a cage;
during mating animals were housed one male to one female;
after mating the males were re-caged as they were before mating; te females were transferred to individual cages
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 28 days (main groups) and 42 days (recovery groups)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C±2 °C
- Humidity (%): 55%±15%
- Air changes (per hr): approximately 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed and suspended in the vehicle. The preparations were made daily (concentrations of 25, 75 and 250 mg/mL) or at weekly interval, in agreement to the stability data. Dosing preparations were kept under magnetic stirring for at least 16 hours at room temperature and up to completion of dosing. Concentrations were calculated and expressed in terms of test item as supplied.

dose volume: 4 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in the range from 25 to 250 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation study (r > 0.99; accuracy 80-120%; precision CV < 10%).

Duration of treatment / exposure:

males: for a minimum of 2 consecutive weeks prior to pairing, through the pairing period and thereafter until the day before necropsy, for a total of 42/43 days.

females: for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum for a period comprised from 51 to 65 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once a week thereafter
- observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals. Animals were examined in an open arena for a minimum of three minutes.

CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during treatment

BODY WEIGHT: Yes
- Time schedule for examinations:
Main groups: Males: weekly from allocation to termination.
Females: weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.
Recovery groups: on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION:
Main groups
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from Day 1 of dosing up to mating. Individual food consumption for mated females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

Recovery groups
The weight of food consumed by each cage of rats was recorded at weekly intervals starting from the first day of dosing

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period, main groups
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters examined: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin,
Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocites, Eosinophils, Basophils, · Monocytes, Large unstained cells), Platelets

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period, main groups
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters examined: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride, Inorganic phosphorus

PLASMA/SERUM HORMONES: Yes (thyroid hormones)
- Time of blood sample collection: at termination (main groups, recovery groups)
- Animals fasted: Yes
- How many animals: all animal

URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of treatment
- 5 males/group from main groups, 5 females of control and high dose from recovery groups)
- Animals fasted: Yes
- Parameters examined: Appearance, Volume (manually recorded), Specific gravity, , pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Blood
sediment: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors, Other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the study, towards the end of treatment
- Dose groups that were examined: all groups, 5 males and 5 females randomly
selected
- Battery of functions tested: sensory activity / grip strength / motor activity


OTHER: for details on reproductive performance, see Iuclid section 7.8

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals, after the mating of all females on Days 43/44
- Maternal animals: All surviving animals, females with live pups were killed on Day 14 post partum
- recovery groups: Animals were killed after 4 weeks of recovery

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

All main group females were examined also for the following:
– number of visible implantation sites
– number of corpora lutea

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the table below were prepared for microscopic examination and weighed, respectively.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups
No treatment-related clinical signs were noted in males and in females of the main groups before the mating period and thereafter during post coitum and post partum periods.
Clinical signs observed at daily intervals were limited to common conditions of the skin and fur (scabs, and/or staining on the body surface, missing of the tail (tip) found in males or females of control and in those receiving 100 or 300mg/kg/day. In any case no more than two animals were affected.

Recovery groups
No clinical signs were recorded in treated animals throughout the study, during treatment and recovery periods.
A subcutaneous mass on the mammary area was observed in one female previously dosed at 1000 mg/kg/day (no. X0164097) on Day 29 of the recovery period. This was, however not considered treatment related.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups
No significant changes in body weight and body weight gain were observed during the study in the treated males, when compared to controls.
Body weight and body weight gain for female animals were generally comparable between the treated and control groups, before mating and during gestation and post-partum periods.
Before pairing, on Day 8, females receiving 1000mg/kg/day showed a decrease in body weight gain (-45%) compared to the control group. This isolated change was followed by a regular growth of the animals and, hence, it was not considered to be toxicologically relevant.
The negative gain weight noted in all groups on the last measurement was due to the fact that the animals were placed under condition of food deprivation for blood collection.

Recovery groups
During treatment or recovery periods, no relevant differences in body weight and body weight gain were noted between control and treated group, of both sexes.
The differences in body weight gain noted in males receiving 1000 mg/kg/day on Day 8 of treatment was considered unrelated to treatment. Infact, on Day 1 of treatment the mean body weight difference between control and treated groups was already evident (467 g. of Group 6 vs 478 g. of control group).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Main groups
Food consumption was unaffected by treatment in both genders during the study.

Recovery groups
During treatment and recovery periods, no differences were noted in food consumption between control and treated group in both sexes receiving 1000 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Main groups:

Haematology: No treatment-related changes were recorded.
The statistically significant changes recorded (erythrocytes, mean corpuscular haemoglobin in females) were not dose-related and/or within the range of expected biological variation, therefore they were considered to be incidental.

Coagulation: No changes were recorded

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Main groups:
No treatment-related changes were recorded.
The statistically significant differences recorded between control and treated animals (urea and potassium in males, cholesterol, calcium and potassium in females) were not dose related and/or within the range of expected biological variation, therefore they were considered to be incidental.

Endocrine findings:

no effects observed

Description (incidence and severity):

The determination of T3, T4 and TSH was performed on:

- Samples from all parental males and females from all main groups


Parental main groups animals:
No treatment-related changes were recorded. T4 was statistically significantly higher than controls in females dosed at 1000mg/kg/day (19%). Since T4 values were within the range of historical data and no other related changes were recorded (i.e. TSH decrease, thyroid weight or liver/thyroid histopathological changes), this finding was considered to be incidental and judged to be due to biological variation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Main groups males and Recovery groups females: No changes were observed.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Main groups
No alterations in motor activity was recorded in any treatment group at the examination performed at the end of treatment. A slight statistically significant increase (+14%) in landing foot splay measurement (first trial) performed toward the end of treatment was recorded in the individual values of Group
4 males (1000mg/kg/day) when compared to control. This change was considered of no toxicological significance since it was observed as isolated case in one sex and associated to the slight positive increase in the mean body weight recorded on the day of the occurrence.

Recovery groups
Motor activity recorded at the end of the treatment period in recovery animals of both gender did not show differences between control and group dosed at 1000 mg/kg/day.
The change (increase) in the motor activity, resulting to be statistically significant,
observed at the end of the recovery period in females previously dosed at 1000 mg/kg/day was considered not treatment-related.
No alterations in grip strength or sensory reaction to stimuliwere observed in any treatment group at the examination performed at the end of treatment or at the end of the recovery period. The statistically significant increase in the grip strength (second trial and mean values) noted at the end of the treatment period in high dose males (1000 mg/kg/day), when compared to the control group was considered a positive effect and then treatment unrelated.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Main groups
Food consumption was unaffected by treatment in both genders during the study.

Recovery groups
During treatment and recovery periods, no differences were noted in food consumption between control and treated group in both sexes receiving 1000 mg/kg/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Main groups:
There were no treatment-related macroscopic observations at the end of the treatment period.
Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.


Recovery groups:
At the end of the recovery period, there were no treatment-related changes following gross pathology examination.
Any observations had a comparable incidence in control and high dose treated groups of both sexes and /or are characteristically seen in untreated Sprague Dawley SD rats of the same age and were considered incidental and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Main groups:
There were no treatment-related microscopic observations at the end of the treatment period.
Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Critical effects observed:

no

Conclusions:

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.

Executive summary:

In a a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD Guideline 422 (adopted on 29 July 2016) Sprague Dawley rats of both sexes were treated with repeated doses of Benzoic acid, C12-15-alkyl esters to assess any general systemic toxic effects of the test item on the animals, as well as possible particular effects on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring related were investigated.

Upon conclusion of the treatment, a 4 week treatment-free period was allowed for groups 5 and 6 in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase.

The vehicle was corn oil. All doses of 100, 300 and 1000 mg/kg/day were administered at a constant volume of 4 mL/kg body weight.

 

Main groups

Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 42/43 days. Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a period comprising from 51 to 65 days.
The following investigations were performed in all groups: mortality check, determination of body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reactivity to stimuli), food consumption, clinical pathology investigations (haematology comprising coagulation, and clinical chemistry in five animals/sex/group randomly selected, urinalysis in males only), macroscopic observations
determination of organweights. In addition, for main phase groups only (parental animals), oestrous cycle evaluation for parental females (2 weeks before dosing, during pre-mating and mating phases, prior to necropsy), assessment of mating performance, thyroid hormone
determination (males and females) and the assessment of litter data were performed. The assessment of clinical signs and measurement of the anogenital distance were performed in all pups. All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum and those sacrificed on Day 14 post partum were subjected to an external examination and the sex was determined by internal gonads inspection.
Thyroid weight and thyroid hormone levels were also determined in 1 pup/sex/group randomly selected at Day 14 post partum.
Routine histopathological examination was performed only in control and high dose groups (five animals/sex/group randomly selected). It included identification of the stages of the spermatogenic cycle in five males.

 

Recovery groups

Recovery males were treated for up to 4 consecutive weeks and killed after 4 further weeks of recovery (treatment free) period.
Recovery females were treated for up to 9 weeks. The females were sacrificed after 4 further weeks of recovery (treatment free) period.
The following parameters were evaluated in the recovery group animals: mortality check, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reactivity to stimuli), body weight and food consumption. Clinical pathology investigations (haematology comprising coagulation, clinical chemistry and urinalysis for females only) at the end of treatment, macroscopic observations and organ weights were also determined.

 

Mortality and fate of females

No mortality occurred throughout the study. The number of females with live pups on Day 14 post partum was 10 of 10 in the control group and in the treated groups at all dose levels.

 

Clinical signs

Main groups and Recovery groups

No treatment-related clinical signs occurred during the study.

 

Neurotoxicity assessment (removal of animals from the home cage and open arena)

Main and recovery groups

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal any changes attributable to the test item.

 

Motor activity, Grip strength and sensory reactivity to stimuli

Main and recovery groups

No treatment-related alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment or recovery period.

 

Body weight and body weight gain

Main groups

No relevant differences were found in body weight and body weight gain between control and treated males and females throughout the study.

 

Recovery groups

During treatment or recovery periods, no differences in body weight and body weight gain were noted between control and treated group of both sexes.

 

Food consumption

Main groups

No effects on food consumption were observed in either males or females during the treatment period.

 

Recovery groups

In recovery animals no relevant changes were noted in food consumption during the treatment or recovery period.

 

Haematology

Main groups

No changes of toxicological significance were recorded in haematological parameters and coagulation.

 

Clinical chemistry

Main groups

No treatment-related changes at clinical chemistry examinations were recorded between control and treated groups.

 

Urinalysis

Main groups males

No treatment-related changes were recorded.

 

Recovery groups females

No treatment-related changes were recorded.

 

Thyroid hormone determination

Adult males and females

No treatment-related changes were recorded in parental males and females.

 

Pups

No relevant changes were recorded in pups of both sexes at Day 14 post partum.

 

Oestrous cycle, reproductive parameters, pairing combination and mating performance

Oestrous cycle, pre-coital intervals and copulatory index did not show any differences between groups. Fertility and copulatory indexes of males and females were similar in all groups.

 

Implantation, pre implantation loss, pre-natal loss data and gestation length of females

Main groups

Gestation periods were similar in treated groups and controls. All pregnant dams gave birth between Days 22 and 23 post coitum. Corpora lutea, number of implantations sites, live litter size, pre-implantation and pre-natal losses of treated groups appeared comparable to control values.

 

Litter data at birth, on Day 1, Day 4 (before culling) and Day 13 post partum of females and sex ratio of pups

Litter data

No relevant differences were observed in litter data between control and treated groups from the day of birth and Day 4 post partum.
After culling litterweight and mean pupsweight (for both sexes combined) of treated groups remained similar to control.
On Day 13 post partum, the statistically significant increase of the presence of male pups in the litters of the group dosed with 1000mg/kg/day matched the decrease of the presence of female pups. A decrease in litter weight and mean body weight of females pups was observed. Only one female pup was present in the litters nos. 65 and 69.

 

Sex ratio

The percentage of males in the group receiving 1000mg/kg/day was slightly higher, when compared to the control values at birth, on Days 4 and 14 post partum. A statistically significant increase in the absolute value of male pups was noted on Day 14 post partum. This increase was balanced by a lower number of female pups, so that a comparable total number of pups (for both sexes combined) between control and treated
groups, was evident.

 

Clinical signs of pups

The pre-weaning clinical signs found in control and treated groups are commonly observed and were comparable between treated and control groups, although an increase in terms of litter affected was noted in Group 4 compared to control. One single pup in two different litters (litter nos. 63 and 71) showed some findings (e.g. small in size and/or apparently no food intake and/or cold to touch) during the first days of lactation. In both cases these pups were found dead or missing shortly after birth. In the other cases the findings were occasionally seen with a trend of recovery (litter nos. 67 and 79). Only in one female (no. 65) more than half of its litter was reduced during the first days after birth in absence of maternal toxicity. It remained unclear whether this observed effect was treatment-related.

 

Anogenital distance and nipple count

No differences in the anogenital distance (normalised value), that can be related to treatment, were noted in pups on Day 1 post partum.

 

Pups thyroid weights

No significant differences were observed at statistical analysis in the weight of thyroid in treated pups, when compared to controls.

 

Necropsy findings in pups and nipple check

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect. No nipples were observed in male pups.

 

Terminal body weight and organ weights

Main and recovery groups

No treatment-related changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls.

 

Macroscopic observations

Main and recovery groups

No remarkable differences were noted at post mortem examination in treated animals, when compared to the controls.

 

Microscopic observations

Main groups

No treatment-related changes were observed in treated animals, when compared to the controls.

 

Conclusion

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.

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 Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test is available for the target substance C12-15 alkylbenzoate.

In addition, a subacute repeated dose toxicity study as well as a subchronic repeated dose toxicity study is available for the source substance Benzoic Acid Isononylester. A justification for read-across is attached to Iuclid section 13.

 

Subacute studies

Study with the target substance

Sprague Dawley rats of both sexes were treated with repeated doses of Benzoic acid, C12-15-alkyl esters to assess any general systemic toxic effects of the test item on the animals, as well as possible particular effects on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring related were investigated.

Upon conclusion of the treatment, a 4 week treatment-free period was allowed for groups 5 and 6 in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase.

The vehicle was corn oil. All doses of 100, 300 and 1000 mg/kg/day were administered at a constant volume of 4 mL/kg body weight.

 

Main groups

Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 42/43 days.

Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a period comprising from 51 to 65 days.

The following investigations were performed in all groups: mortality check, determination of body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reactivity to stimuli), food consumption, clinical pathology investigations (haematology comprising coagulation, and clinical chemistry in five animals/sex/group randomly selected, urinalysis in males only), macroscopic observations, determination of organ weights. In addition, for main phase groups only (parental animals), oestrous cycle evaluation for parental females (2 weeks before dosing, during pre-mating and mating phases, prior to necropsy), assessment of mating performance, thyroid hormone determination (males and females) and assessment of litter data were performed.

The assessment of clinical signs and measurement of the anogenital distance were performed in all pups. All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum and those sacrificed on Day 14 post partum were subjected to an external examination and the sex was determined by internal gonads inspection.

Thyroid weight and thyroid hormone levels were also determined in 1 pup/sex/group randomly selected at Day 14 post partum. Routine histopathological examination was performed only in control and high dose groups (five animals/sex/group randomly selected). It included identification of the stages of the spermatogenic cycle in five males.

 

Recovery groups

Recovery males were treated for up to 4 consecutive weeks and killed after further 4 weeks of recovery (treatment free) period. Recovery females were treated for up to 9 weeks. The females were sacrificed after further 4 weeks of recovery (treatment free) period.

 

The following parameters were evaluated in the recovery group animals: mortality check, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reactivity to stimuli), body weight and food consumption. Clinical pathology investigations (haematology comprising coagulation, clinical chemistry and urinalysis for females only) at the end of treatment, macroscopic observations and organ weights were also determined.

 

No mortality occurred throughout the study. The number of females with live pups on Day 14 post partum was 10 of 10 in the control group and in the treated groups at all dose levels.

No treatment-related clinical signs occurred during the study in main and recovery groups.

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal any changes attributable to the test item in main and recovery groups.

No treatment-related alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment or recovery period in main and recovery groups.

 

No relevant differences were found in body weight and body weight gain between control and treated males and females throughout the study. During treatment or recovery periods, no differences in body weight and body weight gain were noted between control and treated group of both sexes.

No effects on food consumption were observed in either males or females during the treatment period in main and recovery groups.

No changes of toxicological significance were recorded in haematological parameters and coagulation.

No treatment-related changes at clinical chemistry examinations were recorded between control and treated groups.

No treatment-related changes were recorded in urinanalysis.

No treatment-related changes in thyroid hormones were recorded in parental males and females.

No treatment-related changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls.

No remarkable macroscopic differences were noted at post mortem examination in treated animals, when compared to the controls.

No treatment-related microscopic changes were observed in treated animals, when compared to the controls.

Reproductive toxicity aspects of this study are addressed in Iuclid section 7.8. (Toxicity to reproduction).

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.

 

Study with the source substance

Benzoic Acid Isononylester, was administered to Sprague-Dawley rats by gavage (5/sex/dose), for a period of twenty-eight consecutive days at dose levels of 0, 15, 150, and 1000 mg/kg bw/d.

There were no deaths during the study. All animals showed normal gains in bodyweight throughout the study period. Bodyweight gain in test animals was similar to that seen in controls.

A statistically significant increase in clotting time (prothrombin) was detected for males treated with 1000 or 150 mg/kg/day when compared with controls. Females for all dose groups and males treated with 15 mg/kg/day were unaffected.

Animals of either sex treated with 1000 mg/kg/day showed an increase in aspartate aminotransferase achieving statistical significance in females. Males treated with 1000 mg/kg/day also showed reductions in plasma triglycerol and cholesterol levels achieving statistical significance for cholesterol only.

A statistically significant increase in liver weight was observed in either sex treated with 1000 mg/kg/day and in males treated with 150 mg/kg/day. Males treated with 1000 mg/kg/day also showed a slight elevation in relative kidney weight but the difference did not achieve statistical significance. No treatment-related changes were observed among animals of either sex from the remaining treatment groups.

 

The following differences detected between treated and control animals were considered not to be toxicologically significant:

 

Clinical Observations

• Animals treated with 1000 mg/kg/day showed transient episodes of increased salivation from Day 15 onwards. Increased salivation of short duration was also observed in one female treated with 150 mg/kg/day on Day 15 only. Excessive salivation is commonly observed following oral administration and considered attributable to an unpleasant tasting or locally irritant test material formulation rather than an indication of systemic toxicity. No clinical signs were detected in males treated with 150 mg/kg/day or in either sex of animals treated with 15 mg/kg/day.

 

Behavioural Assessments

• Increased salivation was detected in one female treated with 1000 mg/kg/day during the open field assessments conducted at Week 2, this correlated with the daily clinical observations and was therefore considered not to be of neurotoxicological importance.

 

Haematology

• Recovery 1000 mg/kg/day males showed reductions in group mean haemoglobin (p<0.05)

and haematocrit (p<0.01) when compared with controls. However, no such reduction was detected following twenty-eight days of treatment and, as such, these minimal reductions were considered to be of no toxicological relevance.

 

Blood Chemistry

• Plasma creatinine levels were elevated for males treated with 1000 mg/kg/day. However, individual values were all within the normal range (males, creat; 0.42-.0.62 mg/dl) and, in the absence of a concomitant effect on blood urea or any other indication of renal obstruction.

 

• Recovery

1000 mg/kg/day females showed a slight but statistically significant reduction in plasma bilirubin levels in comparison with the corresponding controls, however no such reduction was detected following twenty-eight days of treatment and, as such, this minimal reduction (p<0.05) was considered to have arisen fortuitously.

 

Necropsy

• No treatment-related macroscopic abnormalities were detected in the animals necropsied on Day 29 or Day 43. Incidental findings were confined to stained fur in the ano/genital region detected in one female treated with 1000 mg/kg/day on Day 29. Such staining

observed in isolation is considered unrelated to treatment.

 

Histopathology

• Heart: Focal myocarditis was observed in several control and treated rats and is a common background entity in laboratory maintained rats. The severity of the condition was never greater than minimal, or one or two foci, and should not be interpreted as being indicative of any ongoing myocardial disease.


• Liver: Scattered mononuclear cell foci were observed in the majority of animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered.


• Spleen: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits.


• Kidneys: Isolated groups of basophilic tubules are frequently encountered in the renal cortex of laboratory maintained rats and have no pathological significance at the severities or frequencies reported in this study. Similarly focal corticomedullary mineralisation is a

commonly observed background condition amongst female rats. Hydronephrosis was also reported for one rat. This is widely considered to be a condition of congenital origin and in any event is does not arise as a primary toxicological event.

• Thyroids: Follicular cell hypertrophy is a highly' variable condition and frequently observed as a spontaneous entity among control rats. The group distribution of the condition in this study is not indicative of a treatment-related effect.


• Lungs: A minimal severity of bronchus associated lymphoid tissue was reported for most animals examined in the study and is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis and accumulations of alveolar macrophages are commonly observed pulmonary changes in laboratory maintained rats of this age and are not suggestive of significant respiratory disease.


• Bone marrow: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study there was no difference between control and treated groups.


• Uterus: Dilatation of the uterine horns is a commonly observed cyclical condition in laboratory maintained female rats.

 

Oral administration of Benzoic Acid Isononylester to rats by gavage for a period of twenty-eight consecutive days resulted in treatment related changes in either sex at a dose level of 1000 mg/kg/day and in males treated with 150 mg/kg/day. There was no evidence of treatment related effects in females treated with 150 mg/kg/day or in either sex treated with 15 mg/kg/day for this reason the "No Observed Effect Level" (NOEL) in females was considered to be 150 mg/kg/day and that of males 15 mg/kg/day.

The treatment-related changes seen in male rats treated with 150 mg/kg/day were confined to renal changes consistent with a well-documented condition known as hydrocarbon nephropathy, which only occurs in male rats and is not indicative of a hazard to human health and a marginal increase in clotting time. These changes are not indicative of a serious adverse health effect as defined by the European Labelling Guide, Commission Directive 2001/59/EC.

 

Subchronic study

The oral toxicity of Benzoic Acid Isononylester, when administered daily to rats over a period of 13 consecutive weeks at dosages of 15, 150 and 300 mg/kg/day followed by a recovery period of 4 weeks, has been investigated.

The following investigations were performed: pre- and post-dose observations, clinical signs, neurotoxicity assessment, motor activity, body weight, food consumption, clinical pathology investigations, necropsy and histopathological examination.

No deaths occurred during the study.

No signs of reaction to treatment were revealed during daily pre- and post-dose observations nor during the weekly functional observation tests.

Neurotoxicity tests and motor activity measurements taken at the end of treatment and recovery did not show changes clearly attributable to the test item.

There were no significant differences in body weights between treatment groups during the study.

There was no adverse effect on food consumption during the study.

No lesions of toxicological significance were detected at the ophthalmic examination performed during week 13 of treatment.

No toxicologically relevant changes were observed at the haematological and clinical chemistry analyses carried out during the study.

No significant alterations in urine parameters were observed at the end of treatment and recovery periods.

No treatment-related difference in terminal body weight was detected between groups at the end of treatment and recovery.

Statistically significant increments in relative and/or absolute liver and kidney weights were detected at the high dose level in both sexes and in mid-dose females. Furthermore, a statistically significant increase in absolute thymus weight was recorded at the high dose level in males. No further treatment-related changes were observed among the remaining organs or animals of either sex from the remaining treatment groups. The changes in liver, kidney and thymus weights were reversed at the end of the treatment free period, when compared to the control groups, indicating a full recovery at the high dose level.

No treatment-related findings were observed at macroscopic examination.

No microscopic changes were observed in the examined tissues/organs, which could be considered

treatment-related.

Based on the results of this study, the dose level of 300 mg/kg/day of the test item was considered to be the No Observed Adverse Effect Level (NOAEL).

 

Conclusion

When comparing the effects observed in the subacute toxicity studies, it is noted, that the target substance Benzoic acid, C12-15-alkyl esters did not cause any treatment related effects at the highest dose level of 1000 mg/kg bw/d. Whereas the source substance Benzoic acid isononylester induced hydrocarbon nephropathy at 150 mg/kg/day, which only occurs in male rats and is not indicative of a hazard to human health, and as well as a marginal increase in clotting time. In addition, hepatocyte enlargement as well as some haematological and biochemical blood changes at dose levels of 1000 mg/kg bw/d (females) or starting at 150 mg/kg bw/d (males) suggest effect on liver integrity and/or function. Since these effects showed a tendency towards reversibility after the treatment free period, the changes were not adverse.

Overall, the studies conducted with the source substance Benzoic acid isononylester can be considered as a conservative worst case. However, since no effects were observed in the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted with the target substance itself, the NOAEL of 1000 mg/kg bw/d obtained in this study is regarded as the most relevant result for the endpoint repeated dose toxicity and will be used as point of departure for hazard assessment.

Justification for classification or non-classification

Based on the available data, the substance is not classified as STOT-RE according to regulation (EC) 1272/2008.