Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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
Justification for study design:
No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.

Test material

Constituent 1
Chemical structure
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:
Test Item1 BMS
Synonyms 4-(4-methylphenylthio)benzophenone
Molecular Formula C10H16O
Purity 99.62%
CAS No 83846-85-9
EC No 281-064-9
Appearance White crystals

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
Vehicle:
olive oil
Details on mating procedure:
Normally, 1:1 (one male to one female) was used in the study. The female was placed with the male until evidence of copulation – presence of sperm in vaginal lavage – was observed each morning during examination. Day 0-1 of pregnancy was defined as the day on which mating evidence is confirmed (sperm is found). In the case of unsuccessful pairing, re-mating of females with proven males was carried out. The duration of gestation was recorded and was calculated from Day 0-1 of pregnancy.
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
Details on study schedule:
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.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Parental animals: Observations and examinations:
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.
Oestrous cyclicity (parental animals):
Oestrous cycles (normally lasting 4-5 days in the rats) were examined throughout the pre-treatment period of the study, every morning. The smears were collected at the same time each day to reduce variability when identifying cycling patterns. Vaginal smears were also examined before necropsy (Day 14 post-partum for dams).
Litter observations:
On Day 4 after birth, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four or five pups per sex. Blood samples were collected from the surplus pups, pooled, and stored for possible determination of T4 levels.
No pups were eliminated when litter size was dropped below the culling target (8 pups/litter).
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: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All animals at all dosage levels survived to the scheduled necropsy without significant visible clinical signs.
Dermal irritation (if dermal study):
not examined
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:
effects observed, non-treatment-related
Description (incidence and severity):
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.
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:
effects observed, non-treatment-related
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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Oestrous cycles were monitored 14 days before treatment. All animals were used in the study and 10 females per group showed evidence of copulation.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
parental systemic toxicity
Effect level:
ca. 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All animals at all dosage levels survived to the scheduled necropsy without significant visible clinical signs.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
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:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Sexual maturation:
not examined
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
The anogenital distance (AGD) of each pup was measured on Postnatal day (PND) 4. Pup body weight was collected on the day the AGD is measured and the AGD was normalized to a measure of pup size, preferably the cube root of body weight. The number of nipples in male pups were counted on PND 13.
Nipple retention in male pups:
not specified
Description (incidence and severity):
the number of nipples per areolae were counted on postnatal Day 13 in male pups
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
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.
Histopathological findings:
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.

Developmental neurotoxicity (F1)

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.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on these results, the no-observed-adverse-effect-level (NOAEL) was 600 mg/kg for parental systemic toxicity and 50 mg/kg for reproductive toxicity.