Sulisobenzone

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sulisobenzone

Cas Number:

4065-45-6

Molecular formula:

C14H12O6S

IUPAC Name:

Sulisobenzone

Test material form:

not specified

Details on test material:

Name: Sulisobenzone
CAS No. 4065-45-6
Molecular Weight: 308.3088 g/mol
Molecular Formula: C14-H12-O6-S
SMILES: c1(cc(c(cc1O)OC)S(=O)(=O)O)C(=O)c1ccccc1

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Source: Animals were procured from a CPCSEA approved Vendor [National Institute of Biosciences, PuneHealth Status: Healthy young adult animals were used for the study. Females were nulliparous and nonpregnant.
Age: 12 - 13 weeks at the start of estrous cycle evaluation.
Acclimatisation: Animals were acclimatised to the test conditions for 7 days prior to test item administration.Animal IdentificationDuring acclimatisation period, all the animals were identified by temporary tail marking with indelible ink and cage cards. Following allocation to the study, each animal was uniquely identified by micro toe pad tattooing and colour coded cage cards labelled with study no., study type, test system, sex, dose, group, animal number, dosing start date, experimental start and completion date. Pups were identified with body marking with permanent non-toxic marker pen.
Husbandry/Housing: Before the animals are brought in, the study room and cages were cleaned and disinfected. During the study, the floor of the experimental room and work tops were swept and mopped with disinfectant solution every day or as on requirement. Cages were cleaned at regular intervals.A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). Cage rotation was carried out weekly during study period except during mating and during gestation and lactation only for females. Pregnant females were caged individually.Sterilized corn cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean.Bedding material of batch No. 4-17 and 817 (Krishana Corncob Industries, Aurangabad)) was used in this study and a copy of report of microbial and chemical contaminants analysed periodically by manufacturer of bedding material are incorporated in the raw data.
Environmental conditions: The room temperature was maintained at 19.60 to 24.70°C the relative humidity was kept between 41.80 to 66.60 %. Artificial light was set to give a cycle of 12 hours light and 12 hours dark. Air changes were about minimum 12 times per hour and filtered adequately.
Diet: A conventional laboratory pelleted diet of batch no. 040817, 041017, 040917 and 040617 from approved supplier (Nutrivet Life Sciences, Pune) was offered ad libitum. The copy of composition, WaterAqua guard filtered drinking water in bottles was offered ad libitum. Samples of the drinking water was subjected periodically to bacteriological tests and to chemical contaminant analysis. The latest test results are included in the raw data.

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item. The details of dose formulation preparation is maintained in raw data.
DIET PREPARATION- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:
VEHICLE- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0, 750, 1000 and 1250 mg/kg bw
- Amount of vehicle (if gavage): 2 mL/100g body weight
- Lot/batch no. (if required): A1708001 and A1703001
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Specificity, Linearity, Precision (%RSD), Accuracy (% Recovery) and Homogeneity were analyze by using HPLC-UV.

Details on mating procedure:

- M/F ratio per cage:one male and one female
- Length of cohabitation: Female were placed with same male until pregnancy occurs or two weeks elapsed.
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:The day of detection of sperm positive vaginal smear was considered as day "0" of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol:

Duration of treatment / exposure:

Males: 48 days (main study)
Females: 63 days (main study)
Male and females of recovery groups: 66 days

Frequency of treatment:

Daily

Duration of test:

48 days (male rats, main study)
Approx 63 days (female rats, main study)
66 days + 14 days of recovery (recovery groups).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

G1 (main study), G1-R (recovery group)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

G2

Doses / concentrations 3

Dose / conc.:

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

Remarks:

G3

Doses / concentrations 4

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

G4 (main study), G4-R (recovery group)

No. of animals per sex per dose:

13 rats per sex per dose level (main study)
5 rats per sex per dose level (recovery groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:- Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. The animals were allocated to the different test groups using the ‘Group Allocati on’ function in the MS Excel Add-in “Daniel’s XL Toolbar” (http://xltoolbox.sourceforge.net/). Individual body weights were considered within ± 20% of the groups mean. Details of the randomization was documented in the raw data.- Other:

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes- Time schedule:twice daily (morning and evening)- Cage side observations checked in table [No.?] were included. morbidity and mortality, throughout the acclimatization and study period.
DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: once a day, preferably at the same time each day considering the peak period of anticipated effects after dosing.
BODY WEIGHT: Yes- Time schedule for examinations:Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), day 4 post-partum and before terminal sacrifice.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time
-weighted averages from the consumption and body weight gain data: Yes
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:
OTHER:Hematology and clinical biochemistry, Organ Weight, Gross Pathology and Histopathology were examined.

Ovaries and uterine content:

Oestrous cyclicity and gestation length

Fetal examinations:

Number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and gross abnormalities.

Statistics:

Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks

Indices:

Pregnancy index, copulation, post-natal loss, and pups survival Index.

Historical control data:

Not specified

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No apparent treatment related clinical signs were observed in any of the animals throughout the treatment period. However, statistically significant increase was noted in urine pool in group G2 in male on week 2 as compared to the G1 control group. In recovery female, statistically significant decrease was noted in rearing in G4-R on pretreatment, and increase in fecal bolus on week 1 as compared to the control recovery group G1-R. Statistically significant increase in urine pool in male G2 group on week 2 as compared to the control group G1. Detailed clinical examinations like home cage observation, handling observation and open field obser vation of all animals were observed to be normal during study period in the main study. The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence considered as incidental and not attributed to the effect of test item administration

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality was observed other than two deaths due to gavage error (one male at 750 and one male and 1000 mg/kg).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight changes were restricted to a significant decrease in % body weight change in the recovery group of male rats treated at 1250 mg/kg from day 1 – 22 as compared to the control group. This effect was considered incidental and therefore not attributed to the test chemical.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food intake was unaffected by treatment.

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):

Changes in haematology included a significant decrease in activated partial thromboplastin time in male rats treated at 1250 mg/kg compared to controls; a significant increase in red blood cell levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; a significant increase in platelet levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; and a significant increase in prothrombin time in the recovery group of male rats treated at 1250 mg/kg compared to controls. The changes in haematology were not consistent between groups and lacked histological correlates. Therefore, the changes in haematology were not considered to be of toxicological importance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Changes in clinical chemistry included decreased creatinine levels in male rats treated at ≥1000 mg/kg compared to controls; increased sodium levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased phosphorus levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased aspartate transaminase levels in the recovery groups of male and female rats treated at 1250 mg/kg compared to controls. The changes in clinical chemistry were not consistent between groups and lacked histological correlates. Therefore, the changes in clinical chemistry were not considered to be of toxicological importance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Changes in motor activity and behaviour were restricted to a significant increase in grip strength in male rats treated at 1000 mg/kg bw/day as compared to control. This effect was considered incidental and therefore not attributed to the test chemical.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no significant effects on either the absolute or the relative weight of the brain, adrenals, heart, liver, kidneys, spleen, thymus, thyroid with parathyroid, testes, or epididymides.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All adult animals were normal externally. Visceral internal fidnings included a case of mild splenic enlargement at 1000 mg/kg and one case of mild testicular shrinkage at 1250 mg/kg.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Liver: multifocal minimal to mild Neutrophil/lymphocyte infiltration (Male: G1:2/5, G4:1/5; Female: G1/5), focal to multifocal mild degeneration (Male: G1:1/5, Female: G4/5), Kidneys: focal to multifocal mild lymphocyte infiltration (Male: G1:2/5, G4:1/5; Female: G1/5, G4:1/5); Lungs: multifocal minimal to mild lymphocyte infiltration (Male: G1:1/5, G4:1/5; Female: G1/5, G4:1/5), Heart: focal to multifocal minimal lymphocyte infiltration (Male: G1:1/5; Female: G4:1/5), Spleen: multifocal mild Extramedullary haematopoiesis (Male: G1:1/5), Adrenals: multifocal mild cytoplasmic vacuolation (male: G1:1/5), accessory adrenocortical tissue (Unilateral: Male: G1:2/5, G4:2/5; Female: G1:1/5), Testes: Male: focal mild seminiferous tubule degeneration (Male: G1: 2/13, G2:2/13, G4: 2/13, G1-R: 1/5); focal minimal to mild retention of mature sperm (Male: G1: 1/13, G2:1/13, G3: 1/13, G4: 1/13); focal minimal to mild sloughing of round spermatid/pachytene spermatocyte (Male: G1: 1/13, G2:1/13). Lesions observed in liver, kidneys, lungs, heart, adrenals, spleen and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relation ship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Hormonal data showed no significant effects on the concentrations of T4 or TSH (male and females), testosterone (males) or oestradiol (females).

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No significant effects on post-implnatation loss was observed.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Details on maternal toxic effects:

Oestrous cyclicity was unaffected by treatment and all female rats showed evidence of copulation after the cohabitation/mating period. All females showed precoital intervals of less than 5 days except for four female rats at 750 mg/kg who showed intervals of more than 5 days. Pregnancy rates were 77, 62, 77 and 77% at 0, 750, 1000 and 1250 mg/kg, respectively. No significant effects were observed on gestation length or litter size. Likewise, no significant effects were observed on the number of live births. Post-implantation loss was also unaffected by treatment.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical biochemistry

clinical signs

dead fetuses

effects on pregnancy duration

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

necropsy findings

organ weights and organ / body weight ratios

pre and post implantation loss

Remarks on result:

other: No effect observed

Maternal abnormalities

Abnormalities:

no effects observed

Localisation:

not specified

Description (incidence and severity):

not specified

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No statistically significant effect on pups weight at birth or on PND13

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No statistically significant effect at birth or at PND 13

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant effects observed.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No statistically significant effect on litter size at birth or PND13

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

No significant effects on post-natal loss were observed in treated rats.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Four pups were cannibalized at the 750 mg/kg treatment group. All other pups at 0, 750, 1000 and 1250 mg/kg were normal externally. The internal examination of the pups revealed no abnormalities attributed to the test chemical.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not specified

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

Remarks on result:

other: No effect observed

Fetal abnormalities

Abnormalities:

no effects observed

Localisation:

other: not specified

Description (incidence and severity):

not specified

Overall developmental toxicity

Developmental effects observed:

no

Treatment related:

not specified

Any other information on results incl. tables

Mortality and Morbidity

Sex: Male

Group	Treatment	Dose (mg/kg b.wt.)	No. of Animals/ Group	Observation During Study Period
G1	Control	0	13	NMM
G2	Low	750	13	01/13 (FD on Day 42)
G3	Mid	1000	13	01/13 (FD on Day 21)
G4	High	1250	13	NMM
G1-R	Control- Recovery	0	5	NMM
G4-R	High- Recovery	1250	5	NMM

 

Sex: Female

Group	Treatment	Dose (mg/kg b.wt.)	No. of Animals/ Group	Observation During Study Period
G1	Control	0	13	NMM
G2	Low	750	13	NMM
G3	Mid	1000	13	NMM
G4	High	1250	13	NMM
G1-R	Control -Recovery	0	5	NMM
G4-R	High- Recovery	1250	5	NMM

Keys:NMM = No mortality and morbidity observed, No.= Number, FD= Found dead after dosing due to gavaging error.

Summary of Days of Conception and Pregnancy Index (%)

 

Group(N)	G1(13)	G2(13)	G3(13)	G4(13)
Dose (mg/kg b.wt.)	0	750	1000	1250
No. of females showed evidence of copulation	13	13	13	13
No. of females concieved between Days 1-5 of cohabitation	13	9	13	13
No. of females concieved after Day 5 of cohabitation	0	4	0	0
Females achieved pregnancy	10	8	10	10
Pregnancy Index (%)	76.92	61.54	76.92	76.92

Key:N= number of dams in a group, No. = Number

Mean Gestational Length and Litter size

 

Group(N)	G1(10)		G2(8)		G3(10)		G4(10)
Dose(mg/kg bwt)	0		750		1000		1250
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Gestation Length	22.30	0.48	23.50	1.60	22.10	0.32	22.10	0.32
Litter size (Total No. of litter size)	10.90	2.18	6.38	4.50	9.90	3.03	9.70	3.62

Keys:SD= Standard Deviation, N= number of dams in a group

Note: Three female was non-pregnant in Group G1, G3, G4 and five females were non-pregnant in G2 group.

Mean Pups Body Weight, Sex Ratio and Gross Observation

 

Group	G1			G2			G3			G4
Dose(mg/kg bwt)	0			750			1000			1250
Mean Pups Weight	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Day 0	6.43	0.92	94	6.54	0.46	44	6.00	0.84	96	6.20	0.59	97
Day 4	9.35	1.21	90	8.99	1.07	38	9.36	1.13	91	9.11	1.28	87
Day 13	22.83	3.34	72	20.42	2.37	29	23.46	2.64	68	23.14	3.03	64
Group(n)	G1(10)			G2(8)			G3(10)			G4(10)
Pups Body Weight gain (%)	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Day 0-Day 4	43.96	13.72	90	33.65	9.39	38	55.96	18.26	91	38.77	9.18	87
Day 0-Day 13	140.09	12.44	72	129.99	11.44	29	153.02	12.23	68	134.85	22.05	64
Group (Number of Litter size)	G1(107)			G2(51)			G3(96)			G4(97)
Sex Ratio at birth (Male/Female)	48/46			20/24			49/47			48/49
Sex Ratio at Day 4 (Male/Female)	40/38			17/21			48/43			44/43
Gross Observations	NAD			NAD			NAD			NAD

Keys:SD= Standard Deviation, N= number of dams with live pups on Day 0, n= number of dams with alive pups on Day 4, NAD = No Abnormality Detected

Applicant's summary and conclusion

Conclusions:

NOAEL for systemic and developmental toxicity was established at 1250 mg/kg bw/day in male and female rats. 

Executive summary:

The chemical was given to 13 rats per sex per dose level at 0, 750, 1000 and 1250 mg/kg bw/day by oral gave. Male rats were treated two weeks before mating and thereafter for a total of 48 dosing days. Female rats were treated two weeks before mating, during mating, during gestation and during lactation, for a total of approx. 63 dosing days. No morbidity was observed during the study period. No mortality was observed other than two deaths due to gavage error (one male at 750 and one male and 1000 mg/kg). Clinical findings were sporadic and of no biological significance. Body weight changes were restricted to a significant decrease in % body weight change in the recovery group of male rats treated at 1250 mg/kg from day 1 – 22 as compared to the control group. As such, no significant body weight effects were observed for female rats during the study period. Food intake was unaffected by treatment. Hormonal data showed no significant effects on the concentrations of T4 or TSH (male and females), testosterone (males) or oestradiol (females). Oestrous cyclicity was unaffected by treatment and all female rats showed evidence of copulation after the cohabitation/mating period. All females showed precoital intervals of less than 5 days except for four female rats at 750 mg/kg who showed intervals of more than 5 days. Pregnancy rates were 77, 62, 77 and 77% at 0, 750, 1000 and 1250 mg/kg, respectively. No significant effects were observed on gestation length or litter size. Likewise, no significant effects were observed on the number of live births, pup survival, pup weight or sex ratio. Four pups were cannibalized at the 750 mg/kg treatment group. All other pups at 0, 750, 1000 and 1250 mg/kg were normal externally. The internal examination of the pups revealed no abnormalities attributed to the test chemical. Microscopic examination of the pups’ thyroid and parathyroid glands at 0 and 1250 mg/kg revealed no abnormal findings. In the adult rats, no significant effects were observed on the absolute or relative weights of the testes or epididymides. Grossly, one rat treated at 1250 mg/kg showed a mild reduction in the size of the testes. Organ weight data was not collected for the prostate and seminal vesicles with coagulating glands. Gross and microscopic examination of ovaries, uterus, cervix with vagina, testes, epididymides, prostate, seminal vesicle with coagulating glands at 0 and 1250 mg/kg revealed no significant effects attributed to the test chemical. In the testes, mild focal seminiferous tubular degeneration was observed in 2/13, 2/13, 0/13 and 2/13 rats at 0, 750, 1000 and 1250 mg/kg, respectively. Retention of mature sperm (minimal or mild) was observed in 1/13 rats in all treatment groups. Likewise, sloughing of round spermatids/pachytene spermatocytes was observed in 1/13 rats in all treatment groups. NOAEL for systemic and developmental toxicity was established at 1250 mg/kg bw/day in male and female rats.