4-(4-methylphenylthio)benzophenone

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(4-methylphenylthio)benzophenone

EC Number:

281-064-9

EC Name:

4-(4-methylphenylthio)benzophenone

Cas Number:

83846-85-9

Molecular formula:

C20H16OS

IUPAC Name:

{4-[(4-methylphenyl)sulfanyl]phenyl}(phenyl)methanone

Specific details on test material used for the study:

Synonyms 4-(4-methylphenylthio)benzophenone
Molecular Formula C10H16O
Purity 99.62%
CAS No 83846-85-9
EC No 281-064-9
Appearance White crystals
Storage Room temperature 20±5°C

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Source: Velaz Czech Republic

Sex:

male/female

Details on test animals or test system and environmental conditions:

50 males + 62 females
Age: at least 12 weeks; female animals were non-pregnant and nulliparous
Animal Health : The health condition of animals was examined by a veterinarian before initiation of the study
Acclimation The animals were acclimated to the condition identical to the condition during the experiment 5 days prior to the start of treatment. The acclimation was according to standard operation procedures.
Housing Condition The animals were housed in plastic cages suspended on stainless steel racks in a room equipped with central air-conditioning. The room temperature was within the range of 22 ± 2°C; relative humidity was within the range of 55 ± 10 %. The light regimen was set to a 12-hour light / 12-hour dark cycle The sanitation was performed according to standard operation procedures.
Diet A laboratory food ssniff (ssniff Spezialdiäten GmbH) was offered without limit during the acclimation and the study periods. The certificate of analysis is included in the raw data.
Water The animals were received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is periodical monitored and recorded; certificate of analysis was included in raw data.
Bedding Lignocel SAFE 3/4, Lufa - ITL GmbH, Germany. The certificate of analysis was included in the raw data.
Animals Identification
Each animal was marked with an ID number. Each cage was affixed with a cage card containing pertinent animal and study information. The animals in cages were marked by a line on the tail with an ink marker.
Number of animals in cage was according period of study:
• Pre-treatment period (14 days) – 5 animals per cage
• Pre-mating (14 days) – 5 animals per cage
• Mating (maximum 14 days) - 1 male/1 female per cage
• Gestation (approximately 22 days) - 1 female per cage
• Post-partum (13 days) - 1 female with offspring per cage
Satellite animals – 5 animals per cage during all the study
Justification for the Choice of Species The Test Guideline OECD 422 is designed for use with the rat. The rats are the standard experimental rodent of choice and recommended by OECD Guideline and in the international validation program the rat was the only species used for the detection of endocrine disrupters
the rat was the only species used.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Frequency & Duration Once per day.
Females were treated during:
• 14-day pre-mating,
• 14-day mating (maximum)
• 22-day gestation (approximately)
• 13-day lactation
Males were treated during:
• 14-day pre-mating
• throughout the pairing period for a total of 56 doses
The animals designated for post-treatment observation (5 animals per sex in control and high groups, respectively) remained untreated for subsequent 14 days.

Vehicle:

olive oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Doses of the test item were prepared daily. The required amount of the test item (according to the dose) was diluted in vehicle (olive oil).
The stability and homogeneity of the test item in the vehicle were determined by GC method. The content of test item in the vehicle was checked once during the study. The test item suspended in vehicle was prepared in required amount and theoretical concentrations: Low dose – 25 mg/mL, Mid dose – 100 mg/mL and High dose – 300 mg/mL.
Preparation of the application solution and specification of used test item are included in the raw data. Results of analyses are included in raw data.

Duration of treatment / exposure:

Males were further dosed until total dosing period of 56 days. Females were kept at 14 days after the last treatment of the last pregnant female.

Frequency of treatment:

once daily, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Group Males Females
Total Number ID Number Total Number ID Number
Control 10 1-10 13 51-54; 56-61
Control satellite 5 11-15 5 64-68
Low 50 mg/kg 10 16-25 13 69-78
Mid 200 mg/kg 10 26-35 13 82-91
High 600 mg/kg 10 36-45 13 95-104
High satellite 600 mg/kg 5 46-50 5 108-112

Control animals:

yes, concurrent vehicle

Details on study design:

The females were screened for normal oestrous cycles (in a 2-week pre-treatment period). Dosing of both sexes began 2 weeks prior to mating. Dosing continued in both sexes during the mating period. Males were further dosed until total dosing period of 56 days. They were then killed.
Daily dosing of the parental females continued throughout pregnancy up to and including, Day 13 post-partum or the day before sacrifice.
Animals in a satellite group scheduled for follow-up observations were not mated. Recovery period to detect delayed occurrence, persistence of, or recovery from toxic effects in males was 14 days after 56-days treatment period; females were kept at 14 days after the last treatment of the last pregnant female.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

All animals were checked for morbidity or mortality at twice daily. The health condition of the animals, behaviour, reaction of the animals to the applied item, their well-beings were monitored and recorded in raw data.
General Clinical Observations
General clinical observations were performed once a day, 2 hours after dosing. Detailed clinical observations were made in all parental animals prior to the first exposure, and at a weekly thereafter. The signs as changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity as well as changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies were recorded.
Functional Observations
The functional observations were conducted in five males and five females, randomly selected from each group included:
- Open field test
- Tail flick test
- Grip-strength test
Open field test was made of a dark polyvinyl plastic with dimensions of 60 x 60 cm surrounded by 25 cm high walls. Each session started by placing the rat in the central area of the maze. Test duration was 10 min. The activity was recorded and analysed using ANY-Maze Videotracking Software.
In males the Tail flick test was made shortly before scheduled kill. In females the test was made once during the last week of lactation, shortly before scheduled kill. The tail flick latency was assessed using a tail-flick-meter. A light sensor located under the tail indicated the withdrawal of the tail. The tail was placed on a level surface, a radiant heat was applied to the tail and the latency of the rat to remove its tail from the heat was recorded. A maximum tail-flick latency of 10 sec was used to minimise tissue damage.
For muscle strength assessment, a grip strength meter was used. To perform the evaluation, the animal was pulled by the tail with regular force. The rat could seize a grid attached to a force transducer, till the animal lost its grip. The test was repeated 3 times per forelimb and the maximum grip force (strength in grams) per trial was included in the statistical analysis.

Sacrifice and pathology:

All test animals were subjected to gross necropsy. Full, detailed gross necropsy included careful examination of external surface of the body, all orifices, and cranial, thoracic and abdominal cavities and their contents. All gross pathological changes were recorded for each animal.
The pups were euthanized on PND 13 and examined for gross abnormalities with particular attention to the reproduction organs. From all adult males and females and one male and female pup (PND 13) from each litter thyroid glands were preserved.
Organs of the reproductive system
The number of implantation site
Vaginal smears
The wet weight of testes and epididymides, prostate and seminal vesicles with coagulating glands as a whole of all male adult animals
The ovaries, testes, epididymides, accessory sex organs, and all organs showing macroscopic lesions of all adult animals, were preserved
In addition, for a least five adult males and females, randomly selected from each group
Wet Weight Histopatology
Liver all gross lesions
Kidneys brain (representative regions including cerebrum, cerebellum and pons)
Adrenals spinal cord
Thymus eye
Spleen stomach
Brain small and large intestines (including Peyer's patches)
Heart liver
kidneys
adrenals
spleen
heart
thymus
trachea
lungs
gonads (testis and ovaries)
accessory sex organs (uterus and cervix, epididymides, prostate, seminal vesicles plus coagulating glands)
vagina
urinary bladder
lymph nodes
Peripheral nerve (sciatic or tibial)
skeletal muscle and bone, with bone marrow
The tissues for histology from all animals were preserved in 10% neutral buffered formalin, testes in Davidson fixative and prepared in the paraffin technical. Histopathological examination was performed after preparation of paraffin sections and haematoxylin-eosin staining.

Other examinations:

Body Weight
Each adult animal was weighted on the first day of dosing, weekly during the treatment period and prior to the scheduled euthanasia.
Specifically:
Female rats – dams were weighted on Days 1, 7, 14 and 20 during pregnancy, and on Day 1 (24 hours of parturition), Day 4 and Day 13 during lactation and on Day 14 prior to necropsy.
Males in four experimental groups and two satellite groups were weighted weekly and on necropsy day.
Females in satellite groups were weighted weekly and on necropsy day.
Live pup weight was recorded within 24 hours of parturition, Day 4 and Day 13 post-partum.
Food Consumption
The food consumption was not observed during the mating period in females as well as males. A sample of the diet was retained until finalisation of the report.
Laboratory Examinations
Blood samples were collected from fasted animals; haematology and clinical chemistry parameters were analysed preferably at the same day as blood was collected. Plasma samples for determination of coagulation test and thyroid hormone levels were stored according relevant condition for later analysis. Blood collection for haematological investigations was performed from the incision on the tail; the blood collection for clinical chemistry (serum) from retrobulbar venoplex was performed under anaesthesia (Isofluran). Blood collection for coagulation tests (citrate plasma) and thyroid hormone (EDTA plasma) was taken from the heart.
The blood collection, processing of the blood and the determination of its haematological and clinical chemistry parameters were performed according to the standard operation procedures.
Urinalysis
The urinalysis was performed in five randomly selected males of each group during the last week of the treatment and in satellite animals using urine volume collected from animals in metabolic cages during 6 hours. Following parameters were evaluated: specific gravity leucocytes, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, colour, clarity and volume for 6 hours.
Haematology
Haematological examination was made in five males randomly selected from each group and all females. The blood collection in males was done at the end of the mating period. The blood collection in females was done at prior to of euthanasia of the animals. In satellite animals 14-days after the last treatment.
Clinical Chemistry
Clinical chemistry examination was obtained from the selected five males and all females of each group.
Blood samples for T4 analysis were taken based on the following schedule:
- from at least two pups per litter on day 4 after birth
- from all dams and at least two pups per litter at termination on Day 13
- from all adult males, at termination
Pup blood was pooled by litter for thyroid hormone analyses. Samples intended for hormone determination were obtained at a comparable time of the day.
The plasma T4 levels were analysed by a solid phase competitive Mouse/Rat ELISA Kit according standard procedure of manufacturer and standard operation procedure. Blood samples from the PND 13 pups and the parental males were assessed.

Statistics:

Individual data (five males and all pregnant females) of clinical chemistry, haematology, body weight, relative weight of organs, results of tail flick test and grip-strength test and T4 levels obtained in the experiment were assessed applying statistical software StatgraphicsTM Centurion. Kruskal-Wallis statistical procedure of multiple comparison was adopted to test the null hypothesis that the medians among the dose groups (control, low, mid, high) are the same. The p-value of 0.05 was considered as the level of statistical significance. In the case of statistically significant outcome the Kruskal-Wallis was followed by Mann-Whitney W test to determine which medians are different from which other. Basic descriptive statistics (mean, SD) are reported as well.
Reproduction/developmental and locomotor activity data were analysed using STATISTICA 7.0 (Statsoft, Inc. Tulsa, OK) software. The data are represented as mean ± S.E.M (and were analysed by one-way analyses of variance (ANOVA) followed by Dunnet’s post-hoc test for a multiple comparison procedure to compare each of a number of treatments with a single control. The p<0.05 value was considered statistically significant.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

All animals at all dosage levels survived to the scheduled necropsy without significant visible clinical signs.

Mortality:

no mortality observed

Description (incidence):

During the study no mortality of animals was recorded.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant differences in body weight among the treated groups of adult male and female rats and their control counterparts, except difference in satellite females of High group on first day of the treatment, were observed. Body weight of adult male as well as female rats increased in time in all experimental groups or sporadic short-time body weight stagnation was observed.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of males and females of all dose groups (including satellite animals) was similar to the controls group during the whole study. Increased physiological demands in pregnant and lactating female rats were evident from individual mean consumption per day as well as from total food consumption per week.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decreased of leukocytes (WBC) in Low dose, lymphocytes (Lym) in Mid and High dose and increased of haematocrit (HTC) in Mid and High dose against Control in males were observed. In females were registered statistically significant differences of WBC between Control, Mid and High dose and Lym and granulocytes (Gra) in High dose. In satellite animals decrease of WBC in males and APTT in females were noticed.
The changes were of small magnitude and was not considered as a toxicologically important. During the study, haematology parameters in both sexes were within or close to the historical control data for this species. No test item related effects on the haematology parameters were observed in this study.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant lower activity of Aspartate aminotransferase (AST), concentration of urea and
higher concentration of chloride in males of High dose when compared to the Control group.
Males of Mid dose had statistically significant increase of glucose (GLU) and albumin (ALB) and decrease of urea against Control group. In satellite males no significant differences of monitored parameters were observed.
Females of Mid and High dose had statistically significant increase of Total cholesterol (CHOL) and females of Mid dose increase of concentration of ALB against Control. In satellite females decrease of glucose were registered.
These changes were sporadic, without the test item dose dependent relationship; they were considered to be a result of intra-individual and inter-individual variability for this species or they have only statistical character. The average values of clinical chemistry parameters in all animals were within or slightly outside the serum historical control data. There were no findings in clinical chemistry parameters which could be definitively attributed to the treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Open Field Test
The locomotor activity was influenced only in High group of satellite males. Locomotor activity of females were significantly different between groups, however Dunnett's post-hoc test did not reveal any significant differences between treated groups and control. No other changes in the motor activity were present in the group of males and satellite females.
Tail flick Test
In comparison with Control groups, the reaction time was not influenced by the administration of the test item. No differences were registered in satellite animals
Grip Strength Test
There is not a significant difference between the Control groups and dose groups in males and females. No differences were registered in satellite animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences were found in males. Only statistically significant change in relative weight of left ovary compared Control in females of Low dose was observed. This change was not considered to be test item related. No significant differences were found in satellite males and females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the study, the animals were subjected to gross pathology examination. During the necropsy, sporadic macroscopic findings were described as follow:
Male ID 19 (Low dose) and ID 28 (Mid dose) had thymus with hemorrargic points; in male ID 31 and ID 32 (Mid dose) markedly dilated stomach were observed. Female ID 78 (Low dose) and ID 87 (Mid dose) had cyst on left ovary. No gross lesion in satellite animals were found.
Described macroscopic findings (being sporadic incidence and without test item dose dependence) were considered not to be test item related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopically observed hemorrhagic points in thymus, histologically, were seen focally as hyperaemic capillaries in thymus (ID 1 M/C 53 Th). Similarly, focally hyperaemic capillaries were seen in the lung parenchyma (ID 53 F/C 468 Lu). The lesions arised during not adequate bleeding. Vessels were filled with fresh blood without surrounding acute or chronic inflammatory signs. Lesions are not related to the toxicological study.
Hyperkeratosis of mucosa epithelium in vagina (ID 58 F/C 563 CU; ID 58 F/C 565 V) can be seen in all species (8). Lesion is not related with the test item in experimental animals.
Focal or diffuse glandular hyperplasia in the uterus (ID 1 F/C 579 U) is occasionally observed spontaneously in adult rats. The lesion is especially common in older rats (2). Lesions are not related with the test item.
Golden brown pigment particles, histologically found in ovarium (ID 52 F/C 454 Ov; ID 97 F/H 664 Ov) and uterus wall (ID 53 F/C 487 U; ID 95 F/H 605 U; ID 97 F/H, 667 U; ID 54 F/C, 518 U; ID 59 F/C, 569 U) may be secondary to haemorrhage, especially in multiparous females at sites of placentation and some are ubiquitous in aging animals (14). Lesions are not related with the test item.
Marked neutrophils infiltration of vaginal epithelium and finding of these cells in the lumen (ID 51 F/C 426 V; ID 60 F/C 575 V) is often observed in early metoestrus of rats (1). Lesions are not related to the toxicological study.
In this study, observed changes are considered to be incidental findings or results of experimental manipulation other than administration of the test item. There were no test item - related alterations in the prevalence, severity or histological character of these incidental found lesions.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroxine Assay
Blood samples from post-natal Day 13 (PND 13) pups, and the adult males including the satellite males were assessed for thyroid hormone levels (T4).
There is no statistically significant difference between Control and the dose groups in males. Statistically significant increase of concentration T4 in males of High doses satellite males against Control were registered. The change was of small magnitude and was not considered as a toxicologically important.
Statistically significant decrease of T4 levels in all doses of males pups and Mid and High dose of females pups on PND 13 against Control were observed. The decrease of T4 concentration was about 16-30 % against Control.
Urinalysis
In the urine of some animals, small amounts of protein, ketones and presence of leukocytes were observed. There are no differences between Control and the dose groups and these findings can be considered close to normal (7). No test item related effect was observed.
Reproduction/Development
Oestrous cycles were monitored 14 days before treatment. All animals were used in the study and 10 females per group showed evidence of copulation.
In summary, the tested item dose dependently influenced litter weight at Day 4 post-partum (PP) in Mid and High group and both, male and female pups from high group, has significantly lower weight at Day 4 PP. Similar findings were seen in 13-day-old pups . Increased retention of areolae was observed in Mid group. No other effects were seen after treatment with tested compound .

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The no-observed-adverse-effect-level (NOAEL) was 600 mg/kg for parental systemic toxicity and 50 mg/kg for reproductive toxicity.

Executive summary:

After consideration of the study results following conclusions were made regarding the test item BMS.

The test item did not cause mortality of animals

The test item did not induce significant clinical findings or behavioural deviations in the experimental animals

The test item had no influence on the body weight of adult male and female rats

The test item had not impacted the food consumption

The test item had no influence on the reaction time and grip strength

The test item had no influence on monitored haematological, clinical chemistry parameters and did not cause changes in urine

The test item caused decrease concentration T4 in F1 offspring at Day 13 post-partum (male pups of all dose and female pups of Mid (200 mg/kg) and High dose (600 mg/kg)

The test item dose dependently influenced litter weight at Day 4 post-partum in Mid (200 mg/kg) and High dose group (600 mg/kg) and both, male and female pups from High group, has significantly lower weight at Day 4 post-partum and Day 13 pups

The test item increased retention of areolae in Mid dose (200 mg/kg)

The test item did not influence relative weight of organs

The test item did not cause histopathological changes on examined organs