4'-methoxyacetophenone

Endpoint summary

Description of key information

The NOAEL for the read across substance p-methoxybenzyl acetate (CAS no. 104-21-2) was determined to be 400 mg/kg bw/d for systemic toxicity after repeated application.

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

Study period:

2016-10-11 to 2017-04-06

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:

July 29, 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD), SPF

Details on species / strain selection:

Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control database. In addition, the rat is a required species in the regulatory guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes (not stated directly but females were determined to have normal estrous cycle)
- Age at study initiation: male: 10 weeks; female: 12 weeks
- Weight at study initiation: male: 353.2–402.3 g; female: 228.0–284.1 g
- Fasting period before study: no
- Housing: Animals per cage: 1 (during the quarantine-acclimation period), 1 (during the dosing period), 1 male and 1 female (during the mating period), 1 female and neonates (during the lactation period); Stainless wire mesh cages, 260W×350D×210H (mm) and Polycarbonate cage 260W×420D×180H (mm)
- Diet: ad libitum, Pelleted rodent chow (Teklad Certified Irradiated Global 18 % Protein Rodent Diet 2918C)
- Water: ad libitum, tap water
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The certificate of feed analysis was provided by the supplier and the results of feed analysis met the allowable standard of the test facility.
Public tap water was filtered and irradiated by ultraviolet light. Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year according to the Regulation of Quality Criteria for Potable Water and Test. The results of water analysis met the allowable standard of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7–24.9
- Humidity (%): 43.9–63.6
- Air changes (per hr): 10 – 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The oral route was chosen to assess the toxicity by oral exposure of the test substance.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed using electronic balances (CP323S, CP423S, BP3100S, ENTRIS423i-1S, Sartorius, Germany) and placed in a container. The test substance was mixed with a small amount of vehicle to dissolve using a magnetic stirrer, and then the vehicle was gradually added to yield the desired concentrations. The dosing formulations were stored in a refrigerator (5.0–6.0 °C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as the vehicle because the test substance was well dissolved in it.
- Concentration in vehicle: 0.1 and 200 mg/mL
- Amount of vehicle: 5 mL/kg
- Lot/batch no. (if required): MKBW9504V (Sigma-Aldrich)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dosing formulations were conducted using a gas chromatography (GC-2010 series, Shimadzu Corp., Japan). Samples were taken three times from the middle of each dosing formulation prior to dosing and analyzed for verification of dose level concentration. The results of dose concentration analyses were determined to be in the range of 97.43–103.20 %. These results were within the acceptable limits (± 15 % of nominal values).

Duration of treatment / exposure:

Males of the main group were dosed for a total of 50 days (prior to mating for 2 weeks, during 2 weeks of mating and 22 days of post-mating).
Males and females of the recovery groups were dosed for 50 days.
Females of the main group were dosed for 2 weeks prior to mating until Postpartum Day 13. Females showing no evidence of parturition signs were dosed until Gestation Day 25.

Frequency of treatment:

once daily

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups: 12
Recovery groups: 6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In previously conducted 2-week repeated oral dose range finding study salivation was observed in both sexes at 800 mg/kg bw/day. An increase of the relative kidney weight was noted in males at 800 mg/kg bw/day. Therefore, the high dose level was selected at 400 mg/kg bw/day. Then, the mid and low dose levels were selected at 100 and 25 mg/kg bw/day, respectively.
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

No positive control was conducted.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: general condition, clinical signs, moribundity, mortality and females also signs of abortion and premature birth

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to dosing, once weekly for the dosing and recovery periods
- Detailed clinical observations included: skin, fur, eyes, mucous membranes, occurrence of secretion and excretion, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern etc.), changes in gait, posture and response to handling, and the presence of clonic or tonic movements, stereotypy (excessive grooming, repetitive circling, etc.) or bizarre behavior (self-mutilation, walking backward, etc.)

BODY WEIGHT: Yes
- Time schedule for examinations: males of main group and males/females of recovery group: just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period and recovery period, the day prior to necropsy and on the day of necropsy (fasted body weights); females of main group: just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, the day prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
Individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes, as part of the detailed neurological examination

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 18 h
- How many animals: 6 females and males from the main groups, animals from the recovery groups
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes, 18 h
- How many animals: 6 females and males from the main groups, animals from the recovery groups
- Parameters checked in table No.2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: two days before necropsy
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, but free access to drinking water
- Parameters checked in table No.3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: few days before necropsy
- Dose groups that were examined: 6 males and 6 females were randomly selected from the main groups in addition to all recovery animals
- Battery of functions tested: pinna reflex, auditory reflex, corneal reflex, pupillary reflex, grip strength test, motor activity

IIMMUNOLOGY: Yes
- Time schedule for examinations: pups: on PND 4 and 13; adults: at termination
- How many animals: at least two pups per litter (if available above culling target), all adult males and dams of the main group
- Dose groups that were examined: all
- Parameters examined: Total Thyroxine (T4) [ug/dL] Method: Chemiluminescent competitive immunoassay

Sacrifice and pathology:

Animals that died during the study period:
Necropsies were conducted on all animals found dead as soon as possible. When not feasible, they were stored under refrigeration until necropsy. Gross pathology findings were recorded and tissue samples were collected for histopathology.

GROSS PATHOLOGY: Yes. All surviving animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia. Complete gross postmortem examinations were conducted on all animals including the external surfaces and internal organs.

Organ weights:
The testis and epididymis of all adult males were weighed.
Six males and six females were randomly selected from the main group animals in addition to all recovery animals for necropsy. The organ weights from organs listed in Table No. 4 were determined.

HISTOPATHOLOGY: Yes. The testis, epididymis and eyes with optic nerves were fixed in Davidson’s fixative. All other tissues were preserved in 10 % neutral buffered formalin. The thyroid from one male and one female pups per litter were preserved in 10% neutral buffered formalin on PND 13. Organs listed in Table No. 5 were prepared for histopathology. Examinations were conducted in six males and six females from the control, low, mid and high groups, for all gross, macroscopic lesions in all animals and in addition all tissues from animals found dead or killed in a moribund condition during the study.

Statistics:

The statistical analysis of this study was conducted using the SAS program (SAS® 9.3, SAS Institute Inc., U.S.A.).
For the data including body weights, food consumption, thyroid hormone value, urine volume, hematology and clinical chemistry parameters, organ weights, sensory reactivity and motor activity, the Bartlett test was conducted to test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was employed on homogeneous data, then if significant (significance level: 0.05), followed by Dunnett’s test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneous data, then if significant (significance level: 0.05), Steel’s test was performed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
For the data of recovery groups, Folded-F test was employed to test homogeneity of variance (significance level: 0.05, two-tailed). Student t-test was employed for homogeneity, but if overruled, Aspin-Welch t-test was applied (significance levels: 0.05 and 0.01, two-tailed).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In the main groups, hematuria was observed in one male at 0 mg/kg bw/day from Day 23 to the end of dosing. This symptom was considered to be incidental. Mass in the chest was observed in three females at 100 mg/kg bw/day from GD 9 to the end of dosing. The mass in the chest was a mammary gland tumor, however, this lesion was not considered to be related to the test substance since there was no dose-dependency and it could occur spontaneously. Soiled perineal region was temporarily observed in one female at 25 mg/kg bw/day. This symptom was considered to be incidental because of a lack of dose dependency. Salivation was observed in two males at 400 mg/kg bw/day from Day 25. Salivation was also observed in four females at 400 mg/kg bw/day from GD 3 to PPD 7. However, salivation was considered to have little toxicological significance since it was caused by physicochemical characteristics.

No clinical signs were observed in males and females of the main and recovery groups in the detailed examinations once a week.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

All males of the main group and all animals of recovery groups survived the duration of the study.
In the main groups, one dam at 0 mg/kg bw/day died during parturition on GD 25. Normal gestation period of rats is 21–23 days. The dam showed prolonged pregnancy and dystocia on GD 25. It was judged that cause of death was prolonged gestation period and big size of fetus on GD 25.
One dam at 100 mg/kg bw/day died on PPD 5. This dam was in poor condition such as soiled perineal region, staining around mouth and lacrimation. Pups of this dam had no milk in the stomach. However, the death was considered to be incidental because there was a lack of dose dependency.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant differences in body weight changes were noted in males and females of the main and recovery groups compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In the main groups, statistically significant increases in food consumption were noted in males at 400 mg/kg bw/day from Day 8 to the end of dosing. Statistically significant increases in food consumption were noted in females at 25 and 400 mg/kg bw/day on Day 14. Statistically significant decreases in females at 400 mg/kg bw/day were noted on PPDs 4 and 13.
In the recovery groups, statistically significant increases in food consumption were noted in males and females at 400 mg/kg bw/day.
However, these statistical significances in food consumption had little toxicological meanings since they were not related to body weight changes.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test substance-related effects on pupillary reflex and corneal reflex were observed in animals of both sexes in the main and recovery groups when compared to the control group.

Haematological findings:

no effects observed

Description (incidence and severity):

No test substance-related adverse effects were observed in any animal in the main and recovery groups.
Other statistical significances were considered not to be test substance-related changes because of small magnitude and the values were within the range of historical reference data.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test substance-related adverse effects were observed in any animal in the main and recovery groups.
Other statistical significances were considered not to be test substance-related changes because of small magnitude and the values were within the range of historical reference data.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the main groups, occult blood and erythrocyte were noted in the urine of one male at 25 mg/kg bw/day. However, it was considered to be incidental and not to be a test substance-related effect because there was no dose dependency.
In the recovery groups, no test substance-related effect was noted in males at 400 mg/kg bw/day.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related effects on auditory reflex and pinna reflex were observed in animals of both sexes in the main and recovery groups when compared to the control group.
In the main groups, there was no test substance-related effect on the grip strength in both sexes when compared to the control group. In spontaneous motor activity, a statistically significant decrease in 20–30 min vertical count was noted in males at 400 mg/kg bw/day. However, this statistical significance had little toxicological significance because it was a transient and temporary change.
In the recovery groups, there were no test substance-related effects on the grip strength and spontaneous motor activity when compared to the control group.

Immunological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related adverse effects in total thyroxine (T4) level in P0 males at 25, 100 and 400 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test substance-related adverse effects were observed in any animal in the main and recovery groups.
Other statistical significances in the absolute and/or relative organ weights were considered not to be test substance-related effects because of small magnitude and/or the values were within the range of historical reference data.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dead animals
One dam at 100 mg/kg bw/day was dead on PPD 5. Enlarged adrenal gland and small spleen were observed at necropsy.
One dam of the control group was dead during labor on GD 25. Macroscopic examination at necropsy revealed yellowish white discoloration of the liver, dilated (uncollapsed) lung with hydrothorax and enlarged adrenal gland.
The effects were not considered to be treatment related.

Surviving animals
Macroscopic examination at necropsy did not reveal treatment-related changes in eithermain or recovery groups.
The masses in each subcutis of the axillary and chest region were observed in two dams at 100 mg/kg bw/day. These corresponded to mammary gland adenoma and mammary gland adenocarcinoma. The tumors were considered to be not related to the test substance since it did not show a dose-response relationship and could occur spontaneously. The other macroscopic findings were considered to be incidental findings within the normal range for delivered dams.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Dead animals
In one dead dam at 100 mg/kg bw/day, mineralization at slight/moderate severity in multiple organs (heart, kidney and stomach) was observed along with chief cell hyperplasia at slight severity in the parathyroid and fibrous osteodystrophy at slight/moderate severity in the femur and sternum. The mineralization was observed in vascular wall or perivascular tissues in the heart and stomach. The deposit of minerals in the kidney was mainly observed in the lumens of renal tubules with tubular necrosis. The series of these findings were generally associated with hypercalcemia and hyperparathyroidism, and they were deemed to be the cause of death. However, the findings were considered to be not related to the test substance since they were observed in a single case at 100 mg/kg bw/day and post-partum hypercalcemia could occur rarely. The enlarged adrenal gland was in concordance with adrenocortical hypertrophy. Small spleen, of which cause was atrophy, was also observed in the thymus. These findings were considered to be stress-related and secondary to exacerbated condition.
In one dead dam of the control group, the discoloration of the liver corresponded to microscopically observed bridging centrilobular necrosis at moderate severity. The enlarged adrenal gland was correlated with adrenocortical necrosis at severe severity. Also, necrosis/inflammatory cell infiltration at moderate severity in the heart and multifocal pulmonary thrombus at moderate severity in the lung were observed. The cause of death of this dam was deemed to be multiple organ failure due to dystocia accompanied by shock, which was associated with prolonged pregnancy.

Surviving animals
Microscopic examination did not reveal treatment-related changes in either main or recovery groups.
All other microscopic findings seen in various organs and tissues were considered to be spontaneous or incidental, and to be unrelated to the test substance.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test substance related effects were observed.

Key result

Critical effects observed:

no

Conclusions:

The NOAEL for the test substance was determined to be 400 mg/kg bw/d for systemic toxicity after repeated application.

Executive summary:

An oral repeated dose toxicity study was conducted according to OECD 422 in rats. The test substance was administered via gavage to groups of 12 male and 12 female rats at concentrations of 0 (control), 25, 100 and 400 mg/kg bw/day. Males of the main group were dosed once daily for a total of 50 days (prior to mating for 2 weeks, during 2 weeks of mating and 22 days of post-mating), and females of the main group were dosed once daily for two weeks prior to mating, throughout gestation and for 13 days after delivery. Also, males and females of the recovery groups (6 animals per sex per group) were dosed for 50 days. All males of the main groups and all animals of the recovery groups survived the duration of the study. One dam at 0 mg/kg bw/day and one dam at 100 mg/kg bw/day were found dead in the main group. Salivation was observed in four females in the main group at 400 mg/kg bw/day and one male in the recovery group at 400 mg/kg bw/day during the dosing period, but it was not considered to have toxicological significance. The mass in the chest was a mammary gland tumor, however, this lesion was not considered to be related to the test substance since there was no dose-dependency and it could occur spontaneously. No test substance-related adverse effects were noted in the results of body weights, food consumption, sensory function, motor activity, urinalysis, hematology, clinical chemistry, organ weights and thyroid hormone analysis in adult animals of the test substance-dosed groups. In one dead dam at 100 mg/kg bw/day, mineralization at slight/moderate severity in multiple organs was observed along with chief cell hyperplasia at slight severity in the parathyroid and fibrous osteodystrophy at slight/moderate severity in the femur and sternum. These findings were associated with hypercalcemia and hyperparathyroidism. However, the findings were considered to be not related to the test substance since they were observed in a single case at 100 mg/kg bw/day and post-partum hypercalcemia occurred rarely. In one dead dam of the control group, multiple organ failure was observed and it was due to dystocia accompanied by shock, which was associated with prolonged pregnancy. Based on the results of this study, the NOAEL of the test substance for systemic toxicity was considered to be 400 mg/kg bw/day for animals of both sexes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Read across from guideline study with a close structural analogue.

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

Repeated dose toxicty

Repeated dose toxicity: oral

For the test substance (CAS 100-06-1) itself are no data on oral repeated dose toxicity available. Therefore, a read across with studies for the close structural analogue p-methoxybenzyl acetate ( CAS 104-21-2) was conducted.

The key oral repeated dose toxicity study was conducted according to OECD 422 in rats (2017). The test substance p-methoxybenzyl acetate was administered via gavage to groups of 12 male and 12 female rats at concentrations of 0 (control), 25, 100 and 400 mg/kg bw/day. Males of the main group were dosed once daily for a total of 50 days (prior to mating for 2 weeks, during 2 weeks of mating and 22 days of post-mating), and females of the main group were dosed once daily for two weeks prior to mating, throughout gestation and for 13 days after delivery. Also, males and females of the recovery groups (6 animals per sex per group) were dosed for 50 days. All males of the main groups and all animals of the recovery groups survived the duration of the study. One dam at 0 mg/kg bw/day and one dam at 100 mg/kg bw/day were found dead in the main group. Salivation was observed in four females in the main group at 400 mg/kg bw/day and one male in the recovery group at 400 mg/kg bw/day during the dosing period, but it was not considered to have toxicological significance. The mass in the chest was a mammary gland tumor, however, this lesion was not considered to be related to the test substance since there was no dose-dependency and it could occur spontaneously. No test substance-related adverse effects were noted in the results of body weights, food consumption, sensory function, motor activity, urinalysis, hematology, clinical chemistry, organ weights and thyroid hormone analysis in adult animals of the test substance-dosed groups. In one dead dam at 100 mg/kg bw/day, mineralization at slight/moderate severity in multiple organs was observed along with chief cell hyperplasia at slight severity in the parathyroid and fibrous osteodystrophy at slight/moderate severity in the femur and sternum. These findings were associated with hypercalcemia and hyperparathyroidism. However, the findings were considered to be not related to the test substance since they were observed in a single case at 100 mg/kg bw/day and post-partum hypercalcemia occurred rarely. In one dead dam of the control group, multiple organ failure was observed and it was due to dystocia accompanied by shock, which was associated with prolonged pregnancy. Based on the results of this study, the NOAEL of the test substance for systemic toxicity was considered to be 400 mg/kg bw/day for animals of both sexes.

A supporting repeated dose study with 2 week of application via gavage was conducted as a dose finding study for a 4-week repeated dose toxicity study in rats (2016). The study was conducted according to OECD Guideline 407. The test substance p-methoxybenzyl acetate was administered to rats at 50, 200 and 800 mg/kg bw/day in addition to a control group (corn oil). Each group consisted of 5 males and 5 females. Salivation was observed in three males and one female at 800 mg/kg bw/day since Day 10. The relative kidney weights were significantly increased in males at 800 mg/kg bw/day compared to the controls. There were no treatment-related differences in body weights, food consumption, hematology, clinical chemistry and necropsy findings. Based on the result of this study, the dose levels for combined repeated dose toxicity study with reproduction/developmental toxicity screening test should be selected at 400 mg/kg bw/day for the high dose level and at 25 mg/kg bw/day for the low dose level.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

Based on available data on repeated dose toxicity, the substance is considered to be not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) 2020/1182.