Sodium 4-vinylbenzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

August 27, 2010 to March 22, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Regarding selected animals, male and female Crl:CD(SD) rats (SPF) bred in Charles River Japan Co., Ltd. (3176 Shinyokohama, KouhokuKu, Yokohama, Kanagawa) were selected.
The reason of selection of the animals is that the animals have been widely used in repeated dose toxicity studies and Reproduction/Developmental Toxicity studies, and a lot of background data on this kind of animal and strain has been accumulated.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Regarding selected animals, male and female Crl:CD(SD) rats (SPF) bred in Charles River Japan Co., Ltd. (3176 Shinyokohama, KouhokuKu, Yokohama, Kanagawa) were selected.

The number of 58 males and 82 females of 8 weeks old were purchased. During 7 days including the day of arrival, the animals were housed for quarantine and acclimatization. After that, a period of 8 days was also set as a preliminary housing period.
During these housing periods, clinical observation was conducted every day, and body weights were measured on the day of arrival and on the last day of quarantine/acclimatization period.
During the preliminary housing period, a detailed observation for behavioral function was conducted one time, and estrous cycles were observed every day in females.
As tested animals for this study, 48 males and 68 females were selected from animals which were judged to be normal based on these observations, and also they had their weight closer to the median of body weight measured on the date of start of administration than other unselected animals (Range of body weights at the time of administration, male: 355 to 428 g, female: 220 to 249 g). Both of male and female rats were 10 weeks old on the day of administration start.

Group allocation and the number of animals
For group allocation, one control group and 3 administration groups were basically set, and 12 males and 12 females were allocated to each group. For the study of recovery from the influences of the test substance administration, 10 females each without copulation were additionally allocated to the control group and 500 mg/kg group as satellite animals. For five males each from the control group and 500 mg/kg group, and 5 females each from the satellite control group and the satellite 500 mg/kg group (Recovery animals), a recovery period of 14 days after the end of administration period was set.

Methods of group allocation
Group allocation was conducted by the method to achieve the least deviations of body weight among groups (appropriate stratification method) 5). At first, a single heavier animal from 48 males and 68 females of animals was randomly allocated to every group in turn in the first allocation step
From the second allocation step, the total body weights of all groups were compared each other, and then a single heavier animal was allocated in turn to the lighter group in terms of the total group weight.

Animal identification
Animals were identified by marking to the tails with an oil felt pen. Housing cages were identified by put a label having an identification number. Identification of individual pups was not performed.
Identification of an animal room, and discrimination from other studies and animals.
The animals were housed in independent animal rooms (male: Room No. 313, female: Room No.314) in a barrier area. During the copulation period, females were moved into the room where male animals were kept. Discrimination from the other study and other animal species was performed by indicating the study number, animal species, and animal numbers on the door of an animal room.

Housing conditions
Housing environment: Environmental conditions of the animal rooms, animal cages, etc., are indicated below. The ranges of actual values of temperature and humidity of the animal rooms are shown in parenthesis “< >”.
Temperature: 23±2°C
Humidity: 55±15%
Light and dark cycle: 12 hour light (8:00 to 20:00) / 12 hour dark (20:00 to 8:00)
Ventilation frequency: 15 to 17 times/hour
Methods of housing of animals into cages:
During a mating period; 1 male and 1 female were kept together until copulation was confirmed (Room No.313).
During parturition and lactation; Dams and pups were kept together.
During other periods; Housed individually
Material of cage, shape, size, and so on
During a mating period; Stainless net cage for group housing (340(W) x 294(D) x 176(H) mm)
After 18 day of pregnancy; Stainless cage for parturition (340(W) x 294(D) x 176(H) mm), bedding was put on the cage floor.
During other periods; Stainless bipartite net cage (170(W) x 294(D) x 176(H) mm/ animal)
Bedding: ALPHAdriTM (Sphepherd Specilty Papers, Inc.)
The analytical data of bedding used in this study were obtained from NP Analytical Laboratories, and it was confirmed that there is no abnormality by comparing to the values guaranteed by the manufacturer, and the data were stored.
Methods of sterilization of housing materials: Housing materials (racks, cages, feeder, water supply nozzles, working carts, trays・bedding, etc,) were sterilized with an autoclave (at about 120°C for more than 15 minutes).

Food
Animals were allowed to free access to food supplied by a feeder. Food was CRF1 pellet type (30KGygamma ray radiation sterilization, Lot No.100706) produced in a Chiba factory of ORIENTAL EAST CO., LTD. (3610 Azikisawa, Itabashiku, Tokyo).
However, animals were starved from the evening of the day before necropsy.
Analytical data of the nutrients and contaminants in the food were obtained from ORIENTAL EAST CO., LTD. and stored. It was confirmed that there is no abnormality for food contaminants by comparing with the allowance values specified in the study protocol.

Drinking water
For drinking water, public drinking water (supplied by the Waterworks Bureau of Hatanoshi,
Kanagawa) was filtrated and then irradiated with UV rays, and supplied by an automatic water supplying apparatus in a way that animals were allowed to free access to drinking water.
The water quality test is periodically conducted for our test facility. For the quality of water used in this study, it was confirmed that there is no abnormality by comparing the tested water quality with items and allowance levels specified in the study protocol in which these items and allowance values come from the Water Supply Act.
The water quality test is entrusted to Hatano Research Institute, Food and Drug Safety Center (7295, Ochiai, Hatanoshi, Kanagawa), and the data obtained were stored.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Water for injection (vehicle) was added to the weighed test substance and stirred to prepare solutions with a concentration of 100 mg/ml, 20 mg/ml, and 4 mg/ml (wt/vol). It is noted that since the test substance contains 16.1 % of water, the weight of the test substance was converted to the weight of anhydride, and such converted weight was used for preparation. The test substance was dissolved into water for injection. The preparations of an administration solution were conducted with not exceeding a period of 8 days, and this period is a period in which the stability of the test substance existing in an administration solution was validated. The prepared administration solutions were stored until the administration days under a light shielding condition at a room temperature, and were used within 8 days.

Vehicle
Name: Water for injection (Japanese Pharmacopoeia grade)
Manufacturer: Ohtsuka Pharmacetical Factory, Inc.
Lot No.: OE82N
Storage condition: Light shielding at a room temperature

Details on mating procedure:

For mating, one male and one female from the same dose groups were paired. A copulation period was set as a period from the evening of the 1 day of the copulation period (15 day of administration) until copulation was observed, but the copulation period should be for at most 14 days.
The confirmation of copulation was performed by observing the presences of vaginal plugs in the vagina and sperms in vaginal smears, and the day of confirmation of these observations was set as 0 day of pregnancy. Copulated females were separated immediately from males.
Days necessitated from the start of pairing to the success of copulation were regarded as days needed for copulation. In addition, from the results of mating, a copulation index [(number of copulation pairs / number of pairs) x 100] was calculated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For validation of the concentration and homogeneity of the test substance in administration solutions, 7 samples were taken from an administration solution for each concentration, and concentration of the test substance for each sample was measured with a high speed liquid chromatography (Agilent Technologies 1090).

Duration of treatment / exposure:

Male: Daily administration was conducted for 14 days before the mating period, 14 days during the mating period, and 14 days after the mating period, totally for 42 days.
Female: Females were daily administered for 14 days before mating, during the mating period (until the success of copulation, for at most 5 days). Females those which mated and delivered were daily administered during the gestation period and 4 days of lactation, totally for 42~46 days. Those females which mated but showed no parturition were daily administered for 41~43 days which is equivalent to 25 days of pregnancy.
However, females on delivering and females which delivered just before administration were not administered on such days. Female of the satellite group were administered daily for 42 days.
For five males and 5 satellite females in the control group and 500 mg/kg group, a recovery period of 14 days was set after the end of administration period.

Frequency of treatment:

Daily administration.

Details on study schedule:

Screening study for reproductive toxicity/developmental toxicity so F1 animals not mated.

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 per treatment group/per sex
Additional 20 females in a satellite group (10 per dose)

Control animals:

yes, concurrent vehicle

Details on study design:

The reason of selection of the animals is that the animals have been widely used in repeated dose toxicity studies and Reproduction/Developmental Toxicity studies, and a lot of background data on this kind of animal and strain has been accumulated.
In order to decide the administration doses for this study, a pilot study of 14day repeated oral administration (hereinafter, pilot study) was conducted. In the pilot study, 1000 mg/kg as the highest dose, 600 mg/kg, 300 mg/kg, and 100 mg/kg were set. 2,4-dimethylbenzenesulfonic acid was administered through oral gavage to 5 male and 5 female Crl:CD (SD) rats in each group once a day for 14 days. Animals were necropsied on the next day after the end of the administration period.
As the results, 1 male of the 1000 mg/kg group and 1 female of the 600 mg/kg group were moribund with dyspnea, and eventually sacrificed in extremis. In these 2 animals, an insufficiency of constriction in the lung was observed without other abnormalities.
Arrhythmic respiration was observed in many males and females of the 1000 mg/kg group in the early stage of administration period. In addition, an abnormal respiratory sound was heard in several animals.
In both sexes in the 1000 mg/kg and 600 mg/kg group, body weight and food consumption were decreased in 1 week of administration, but a tendency of recovery from such decrease was noted in 2 week of administration. In the result of necropsy, thickening of the stomach walls was observed in several cases in both sexes in the 1000 mg/kg and 600 mg/kg group.
No influences due to administration were noted in both sexes of the 300 mg/kg group and 100 mg/kg group.
From the above results, it was judged that the highest dose for this study would be set at near 600 mg/kg at which a moribund animal was observed, rather than at 300 mg/kg at which no influence was noted. Therefore, the highest dose of 500 mg/kg, middle dose of 100 mg/kg, and low dose of 20 mg/kg were set.

Positive control:

Positive control not required for the study type.

Parental animals: Observations and examinations:

Clinical observation
Clinical observation was conducted twice a day (before and after of administration) during the administration period, and once a day (in the morning) during the recovery period.

Function observational battery (FOB)
1) Detailed observation
Detailed observation of behavioral function was conducted at the time of taking out animals from cages, after taking out, and putting on a working table. At the time of taking out from cages, a touch response and easiness of handling were observed. After taking out from cages, an easiness of handling, body temperature, fur condition, color of the skin, conditions of eyes, and existences of salivation, urination, and of defecation were observed.
On a working table, animals were checked for posture, activity, alert/exploratory behavior, gait, stereotypic behavior, respiration, and existence of tremor, twitching, and of convulsion.
2) Response tests
Response tests were performed on visibility, auditory sense, pain sense, pupillary reflex, and midair righting reflex.
3) Measurement of grip strength
Grip strengths of the forelimb and hindlimb were measured twice each using a Dynamometer (MK380CM, Muromachi Kikai Co., Ltd.).
A mean value was calculated for each animal.
4) Assessment of locomotor activity
Locomotor activity during 60 minutes in each animal was measured using a locomotor activity detector (SCANET MV10, MELQUEST Ltd.).
5) Frequency of locomotor activity observation
Male: Detailed observation was conducted once a week during the administration period and recovery period. A response test, grip strength measurement, and locomotor activity measurement were conducted once in the 6th week of administration.
Female: Detailed observation was conducted once a week during the administration period and recovery period. However, regarding the observation in the 6th week of administration, for the reason for reducing the stress on dams during parturition and lactation, only satellite animals were observed. For the parturition animals, each 5 animals whose delivery date was close to the measurement day, which was the day before necropsy, were selected from each group, and conducted the response test, measurement of grip strength, and locomotor activity measurement on the measurement day. Satellite animals were tested once in the 6th week of administration.

Body weighs
Male: Body weights were weighed on 1 day (starting day of administration), 8, 15, 22, 29, 36, and 42 day of administration during the administration period, and 1, 8, and 14 day of recovery during the recovery period.
Females for mating: Body weights were weighed on 1, 8 and 15 day of administration before mating. After the mating, pregnant females were weighed on 0, 7, 14 and 20 day of pregnancy, and females after parturition were weighed on the day of parturition and 4th day after parturition (0 day of lactation and 4 days of lactation).
Satellite females: Body weights were weighed on 1, 8, 15, 22, 29, 36, and 42 day of administration during the administration period.
During the recovery period, body weights were weighed on 1, 8, and 14 day of recovery.
On the day of necropsy, body weights after starvation (body weights at carrying out) were weighed. Dead animals were weighed at the time of carrying out.

Food consumption
Male: During the administration period, food consumptions were measured for periodical consumptions of these periods of 1~8 day, 8~15 day, 29~26 day, and 36~42 day of administration. During the recovery period, food consumptions were measured for the periodical consumptions of these periods of 1~8 day, and 8~14 day of recovery.
Female for mating: Food consumptions were measured for periodical consumptions of these periods of 1~8 day, and 8~15 days of administration before mating.
After the mating, food consumptions of pregnant females were measured for periodical consumptions of these periods of 0~7 day, 7~14 day, and 14~20 day of pregnancy. For females after parturition, food consumption was measured for consumption of the period of 0~4 day of lactation.
Satellite females: During the administration period, food consumptions were measured for periodical consumptions of these periods of 1~8 day, 8~15 day, 29~26 day, and 36~42 day of administration. During the recovery period, food consumptions were measured for the periodical consumptions of these periods of 1~8 day, and 8~14 day of recovery.

Urinalysis
For males and satellite females, fresh urine samples were collected before administration once in the 6th week of administration, and the following test items were examined with a test paper (Multistix, Siemens Healthcare Diagnostic).
Test items: pH, protein, glucose, ketone bodies, bilirubin, occult blood, urobilinogen
One female from the satellite group of 500 mg/kg was positive in glucose test, and therefore this animal was examined for glucose at the end of recovery period.

Necropsy
Male animals except animals for recovery test were necropsied on the next day of the last day (42 day) of administration. (Necropsy at the end of the administration period). Animals for recovery test were necropsied on the next day of the end of the recovery period (a period of 2 weeks after the end of administration) (Necropsy at the end of the recovery period).
Females for mating were necropsied on the following days.
Females with parturition: 5 days after parturition
Females which mated but did not show parturition: the day equivalent to 26 day of pregnancy.
Satellite females except animals for recovery test were necropsied on the next day of the last administration (Necropsy at the end of the administration period).
Animals of recovery test were necropsied on the next day after the end of the recovery period (Necropsy at the end of the recovery period).
Above animals for necropsy were anesthetized with ether to sample blood from the abdominal aorta, and then exsanguinated to death.
Dead animals were necropsied as soon as possible after the discovery of death.

Hematology
Animals survived on the day of necropsy were anesthetized with ether to sample blood from the abdominal aorta, and the following test items were examined. For hematology, whole blood collected into a tube containing EDTA2 potassium was used, and plasma obtained by centrifugation from the blood collected into a tube containing sodium citrate was used (for items marked with “*”).
Test items: Erythrocyte count, hemoglobin concentration, hematocrit, leucocyte count, differential leukocyte count, platelet count, prothrombin time (PT)*, activated partial thromboplastin time (APTT)*, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte ratio.

Blood biochemistry
Animals survived on the day of necropsy were anesthetized with ether to sample blood from the abdominal aorta and to collect into a tube containing heparin lithium. For the examination of the following test items, plasma obtained by centrifugation was used.
Test items: Total protein, albumin, A/G ratio, glucose, total cholesterol, triglyceride, phospholipids, blood urea nitrogen (BUN), creatinine, sodium, potassium, chloride, calcium, inorganic phosphorus, AST, ALT, ALP, γGTP, LDH, total bilirubin, CK.

Hormone analysis in blood
Animals survived on the day of necropsy were anesthetized with ether to sample blood from the abdominal aorta and to collect into a tube containing heparin lithium.
For the examination of the following test items, plasma obtained by centrifugation was used for the examination of triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) concentrations.

Parturition and lactation
Those females mated were transferred to the cages with bedding material for delivery on 18 day of pregnancy. The confirmation of the end of delivery was conducted twice a day from 20 day of pregnancy. If the delivery was finished until 16:00, such day was regarded as the delivery day (0 day of lactation). If animals being in the middle of delivering were seen, the condition of delivery was observed. The conditions of lactation were observed daily from 0 day to 4 day of lactation.
A period from 0 day of pregnancy to the day of delivery was regarded as a gestation period. A fertility index [(number of pregnant female / number of copulating pairs) x 100] and a gestation index [(number of females delivering live pups / number of pregnant females) x 100] were calculated.
Females which did not deliver until equivalent 25 day of pregnancy were necropsied on the next day to examine if they were gestation.

Number of corpora lutea of pregnancy and implantation sites
The ovaries of mated females were excised out at the time of necropsy, and the number of corpora lutea of pregnancy were counted under a stereoscopic microscope. Further, the uteri were excised out to count implantation sites.
From these results, an implantation index [(number of implantation sites/number of corpora lutea of pregnancy) x 100] was calculated.
Females which did not show parturition nor implantation sites were regarded as an infertile animal.

Oestrous cyclicity (parental animals):

In the every morning from the starting day of administration to the first day of mating, vaginal smears of female animals (excluding satellite animals) were taken and Giemsa-stained slides were prepared. These slides were observed under a light microscope for an estrous cycle.
An estrous cycle was classified as proestrus, estrus, and diestrus. The animals which showed repeating estrus phase within 4 to 7 days were judged to be normal. In addition, a mean duration of an estrus cycle was calculated for animals showing one or more estrous cycles.

Sperm parameters (parental animals):

Not examined as part of this study.

Litter observations:

Number of pups delivered, sex and external observation
On the day of delivery (0 day of lactation), the number of delivered pups (live born pups and dead pups) was counted. Also the confirmation of sex and the external observation of live pups were conducted.
From these results, a delivery index = [(Number of delivered pups /number of implantation sites) x 100], live birth index = [(number of live pups on 0 day of lactation / number of implantation sites) x 100], viability index on 0 day of lactation = [(number of live pups on 0 day of lactation) / number of delivered pups] x 100], sex ratio of born pups (number of delivered male pups / number of delivered male and female pups), sex ratio of live pups ( number of live male pups / number of live male and female pups) were calculated.

Number of live pups and clinical observation
On every day until 4 day of lactation, the number of live pups was counted, and clinical observation was conducted.
From the result, a viability index on 4 day of lactation [(number of live pups on 4 day of lactation / number of live pups on 0 day of lactation) x 100], and a sex ratio of live pups on 4 day of lactation (number of live male pups on 4 day of lactation / number of live male and female pups on 4 day of lactation) were calculated.

Body weight
For pups living on 0 day and 4 day of lactation, firstly a total body weight for male pups or female pups per maternal animal (per litter) was measured, and then a mean body weight per pup was calculated.

Postmortem examinations (parental animals):

Pathological examination
1) Necropsy
All animals were examined for the presence of macroscopic pathological changes.
2) Organ weight
Wet organ weights (absolute organ weights) were weighed in the following organs of animals survived at the time of necropsy. Wet organ weights of pituitary, thyroid, prostate (ventral lobe), and seminal vesicle with coagulating gland were weighed after one night fixation in 10% neutral buffered formalin solution. In addition, a percentage of an absolute organ weight to a final body weight (a relative organ weight to a body weight) was calculated.
Weighed Organs: Pituitary, thyroids, heart, liver, kidneys, thymus, spleen, adrenal gland, testes or ovary, epididymides or uterus, prostate (ventral lobe), seminal vesicle with coagulation gland, brain, lungs.
3) Excision of organs and preservation
The following organs/tissues were excised out, fixed in 10% neutral phosphate buffered formalin solution and preserved. The testes and epididymides were fixed in Bouin solution and then preserved in 10% neutral buffered formalin solution.
Preserved organs and tissue: Brain, pituitary, spinal cord, eyes, Harderian glands, thyroids, parathyroid, heart, tongue, salivary glands, esophagus, nasal cavity (fixative solution was injected into the nasal cavity), nasopharynx, larynx, trachea, lung (left lobe was injected with fixative), liver, kidneys, thymus, spleen, adrenal glands, pancreas, stomach, small intestine (include duodenum), large intestine, testes, epididymides, seminal vesicle・coagulating glands, prostate, ovaries, uterus, vagina, urinary bladder, lymph nodes (axillary, inguinal and others), peripheral nerve (sciatic nerve), bone marrow (femoral), skin, mammary glands, muscle.
4) Histopathology
Histopathology examination of organs indicated below was conducted in all animals. The fixed organs and tissue were embedded in paraffin, thin sectioned, and stained with hematoxylin and eosin. These histological slides were observed under a light microscope. For the kidneys of animals showing eosinophilic bodies in the examination of kidneys, immunohistological staining method for α2u globulin was conducted for such liver.
Examined organs and tissue: Brain, pituitary, spinal cord, eyes, thyroids, parathyroids, heart, nose, trchea, lung, liver, kidneys, thymus, spleen, adrenal glands, stomach, small intestine (include duodenum), large intestine, testes, epididymides, seminal vesicle・coagulating glands, prostate (ventral lobe), ovaries, uterus, vagina, urinary bladder, lymph nodes (axillary, inguinal and others), peripheral nerve (sciatic nerve), bone marrow (femoral), mammary glands, and muscle.

Postmortem examinations (offspring):

Pups living on 4 day of lactation were sacrificed by exsanguinations from abdominal aorta under anesthesia with ether in order to be necropsied. Organs in the thoracic cavity and abdominal cavity were excised out, and fixed in 10 % buffered formalin solution. Organs excised from pups delivered from the same maternal animal (litter) were preserved all together. In addition, the remained parts of carcasses were fixed and preserved in ethanol.
For pups died before 4 day of lactation, organs in the thoracic cavity and in the abdominal cavity were discarded after necropsy, and the remained parts of carcasses fixed and preserved in ethanol.

Statistics:

The effective number of animals was the number of animals used in each group.
In all examinations and measurement, the numbers of animals which were subjected to these examinations and measurement were regarded as the number of samples.
The results of copulation index, fertility index, gestation index, urinalysis, and histopathology were analyzed with x2 test between the control group and each dose group.
For numerical data obtained in the other examinations, the preliminary test for homogeneity of variances was conducted in Bartlett’s test in a way that the control group was set as a standard group.
As a result, if a variance was shown to be homogenous, a one-way analysis of variance was conducted, and in the case that there was a significant difference between groups, a mean value was analyzed using Dunnett’s test for multiple comparison. If a variance was shown to be unequal, data of each group were transformed to ranks and analyzed in a Kruskal-Wallis rank sum test, and in the case that there was a significant difference between groups, the data were analyzed using Dunnett’s test for multiple comparison.
The numerical data of satellite female groups and the numerical data obtained during the recovery period in the male recovery group were firstly analyzed in an F-test. If there was no difference in distribution, a Studen’s t-test was applied. If there was difference in distribution, an Aspin-Welch’s t-test was applied.
In each statistical test, a two sided test at the significance level of 5% and 1% was applied to report the results.

Reproductive indices:

For an estrous cycle, the duration from the next day of estrus to the end of the next estrus was regarded as 1 estrous cycle, and a mean value was calculated from all cycles observed during an observation period. The mean value was indicated to one decimal place by rounding.
Mean values and standard deviations for each group for days needed for copulation, gestation period, number of corpora lutea of pregnancy, number of implantation sites, number of delivered pups, and number of live pups, were expressed to one decimal place by rounding.
A copulation index, fertility index, gestation index, implantation index, deliver index, live birth index, and viability index of 0 day of lactation were expressed to one decimal place by rounding off.
A sex ratio was expressed to two decimal places by rounding.

Offspring viability indices:

A body weight per pup was weighed in gram. Total body weights for male pups and female pops was weighed all together for each sex to one decimal place. The Body weight per pup was calculated by dividing the total body weight for each sex with the number of measured pups, and it was expressed to one decimal place by rounding.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Male: Abnormal respiratory sounds were heard in 7 animals in the 500 mg/kg group. This finding was observed only once or intermittently before and after daily administration. One of these animals showed irregular respiration.
One animal (Animal No. 1309) of recovery animals in the 500 mg/kg group showed deep respiration on 8 day of recovery. This finding was continuously observed until the end of the recovery period. In this animal, decrease in the volume of feces and contamination around the external reproductive organ with urine were observed, and finally hypothermia was also observed.
No influences of administration were noted in the 20 mg/kg group and 100 mg/kg group in the clinical observation.
Female: One of satellite females in the 500 mg/kg group died on 10 day of administration. This animal showed no abnormal conditions before death and no external abnormalities at the time of death.
During the administration period, in the 500 mg/kg group, abnormal respiratory sounds were heard in total 5 females which were 3 copulating females and 2 satellite females.
These findings were observed only once or intermittently before and after daily the administration. Among these animals, one copulating female showed open mouth respiration, irregular respiration, bloody discharge attachment around the nostril, and abdominal distention. In this animal, decrease in locomotion activity and contamination around the external reproductive organ with urine were also observed. However, these abnormal findings disappeared several days later. In addition, open mouth respiration and irregular respiration were observed in one of satellite females, but recovered within 2 days. One animal out of 3 remaining animals showed temporal deep respiration and bloody discharge attachment around the nostril.
In the 500 mg/kg group, contamination around the external reproductive organs with urine was observed besides abnormal respiration. This finding was temporarily observed in 3 copulating females and 3 satellite females.
In addition to the above findings, localized alopecia was observed in each one animal in the 20 mg/kg, 100 mg/kg and 500 mg/kg group. However, since this finding showed no dose dependency, it was considered to be an incidental symptom unrelated to the administration.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One of satellite females in the 500 mg/kg group died on 10 day of administration. This animal showed no abnormal conditions before death and no external abnormalities at the time of death

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male: The body weights of the 500 mg/kg group were lower during the administration period and the recovery period compared to those of the control group (no statistical significance). The body weight of one animal (Animal No. 1309) of the 500 mg/kg group, which showed abnormal respiration during the recovery period, was decreased gradually after 36 day of administration, and from 8 day of recovery on which abnormal respiration was observed, it was further decreased severely.
No influences of administration were noted in the body weight changes in the 20 mg/kg and 100 mg/kg group.
Female: No influences of administration were noted in the body weight change of copulating females.
The body weight of satellite females in the 500 mg/kg showed a statistical significant low level on 29 day of administration. However, since no differences of body weights were observed on the other days, it was judged that there was no suppression of increase of the body weight during the administration period. Accordingly, no influences of administration were noted in the body weight change of satellite female animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male: Food consumption during 1 week of administration in the 500 mg/kg group was lower compared to that in the control group (no statistical significant difference). Thereafter, there were no differences in food consumption compared to that of control group during the administration period. The food consumption of one male (animal No. 1309), which showed abnormal respiration during the recovery period, was severely decreased in 2 week of recovery.
No influences of administration were noted in the 20 mg/kg group and 100 mg/kg group.
Female: No influences of administration were noted in copulating females and in satellite females during the administration period.
In satellite females of the 500 mg/kg, food consumptions during 1 and 2 week of recovery showed higher values compared to that in the control group (no statistical significance in 2 week of recovery).

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):

Male: Decrease in MCH was noted in the 500 mg/kg group at the end of the administration period. This finding was not noted at the end of the recovery period.
Hemoglobin concentration of the 100 mg/kg group was higher compared to that in the control group. However, since no difference was noted in the 500 mg/kg group compared to the control group, the finding was considered not to be the influence of administration. Accordingly, influences of administration were not noted in the 20 mg/kg group and 100 mg/kg group in the hematological examination.
Female: In the 20 mg/kg group and 100 mg/kg group, PT was higher than that of the control group, and an unclassified leukocyte ratio (indicated in “others”) was lower than that in the control group. However, since none of these findings were noted in the 500 mg/kg group, these findings were judged not to be related to the administration. Accordingly, no influences of administration were noted in pregnant females and parturition females in the hematological examination.
APTT of satellite females of the 500 mg/kg group was lower than that of the control group at the end of the administration period. However, this finding was not noted in pregnant and parturition females, and the degree of this change was slight, which was disappeared at the end of the recovery period, and consequently this finding was judged not to be the influence of administration. Accordingly, no influences of administration were noted in satellite females in the hematological examination.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Male: Decreases in total protein and albumin levels were noted in the 500 mg/kg group at the end of the administration period. Both findings were not observed at the end of the recovery period.
In one animal (animal No. 1309) of the 500 mg/kg group, which showed abnormal respiration during the recovery period, ALT and AST values were higher compared to other animals (ALT 361 IU/L, AST 383 IU/L).
No influences of administration were noted in the 20 mg/kg group and 100 mg/kg group in the blood biochemical test.
Female: Decreases in the levels of BUN, creatinine, and inorganic phosphate were noted in pregnant and parturition females of the 500 mg/kg group. Decrease in the level of total protein and elevation of AST level were noted in satellite females of the 500 mg/kg group at the end of the administration period. These findings were not observed in satellite females of the 500 mg/kg at the end of the recovery period.
At the end of the recovery period, the level of inorganic phosphate in satellite females of the 500 mg/kg group was higher than that in the control group. However, the finding was not noted in satellite females and in pregnant and parturition females at the end of the administration period, and the degree of the finding was slight, and consequently this finding was judged to be unrelated to the administration.
Increase in the level of total bilirubin was noted in the 20 mg/kg group. However, since the finding was a slight change without a dose dependency, this finding was judged to be unrelated to the influence of administration. Accordingly, no influences were noted in the 20 mg/kg group and 100 mg/kg group in the blood biochemical test.

Hormone analysis in blood
Male: No influences of administration were noted on the levels of plasma T3, T4, and TSH concentration in blood.
Female: No influences of administration were noted on the levels of plasma T3, T4, and TSH concentration in blood.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Male: No influences of administration were noted in the results of urinalysis conducted in 6 week of administration.
Female: In the results of urinalysis conducted for satellite females in 6 week of administration, one animal (animal No. 2508) of the 500 mg/kg group showed positive (3+) reaction to glucose. After the end of the recovery period, when the presence of glucose in urine was analyzed again in this animal, the result was negative.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Findings in detailed observation
Male: In the detailed observation of behavioral function during the administration period, in the 500 mg/kg group, abnormal respiratory sound was heard in 4 animals, irregular respiration was observed in 2 animals, and contamination around the nostril was observed in one animal. In addition, during the recovery period, one animal of the 500 mg/kg group showed deep respiration, contamination around external reproductive organ, and hypothermia.
No influences of administration were noted in any items of observation in the 20 mg/kg group and 100 mg/kg group.
Female: In the detailed observation of behavioral function during the administration period, in the 500 mg/kg group, abnormal respiratory sound was heard in one animal, and contamination around external reproductive organ was observed in other one animal. No abnormalities were observed in satellite females during the recovery period.
No influences of administration were noted in any items of observation in the 20 mg/kg group and 100 mg/kg group.

Response
Male: In the response test conducted in 6 week of administration, no influences of administration were noted in any items of observation.
Female: In the response test where copulating females were examined 4 day after delivery (the day before necropsy), and satellite females were examined in 6 week of administration, No influences of administration were noted in any items of observation.

Grip strength
Male: In the grip strength test conducted in 6 week of administration, no influences of administration were noted in the grip strengths of forelimb and hind limb.
Female: In the grip strength tests where copulating females were examined 4 days after delivery (on the day before necropsy) and satellite females were examined in 6 week of administration, No influences of administration were noted in the grip strength of forelimb and hind limb.

Locomotor activity
Male: In the test of locomotor activity conducted in 6 week of administration, no influences of administration were noted.
Female: For females (dams) measured 4 days after delivery (on the day before necropsy), the locomotor activities of the 100 mg/kg group and 500 mg/kg group during a period of 10 minutes from the test duration time of 20 to 30 minutes were low with a statistically significance. However, no statistical significance was noted in the total locomotor activity during a whole testing duration (60 minutes). Also no influences of administration were noted in the locomotor activity of satellite females of the 500 mg/kg group tested in 6 week of administration. Therefore, it was judged that there were no influences of administration on the locomotor activity in female.

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):

Male: In all tissues examined, there were no pathologic lesions related to the influence of administration.
Since abnormal respiration was observed in the clinical observation, the histopathological examination of the nose for all cases was conducted, but no abnormalities were observed.
Atrophy of seminiferous tubules in the testes was observed in one animal of the 100 mg/kg group at the end of the administration period, and in one animal of the 500 mg/kg group at the end of the recovery period.
However, since these lesions were slight or moderate lesions which were observed on the unilateral side, the lesions were judged not to be the influence of administration.
In addition, the testes of all animals were scrutinized for a stage cycle of spermatogenesis, but no abnormalities were observed.
Eosinophilic bodies in the renal tubular epithelia were observed in each one animal at the end of administration period and recovery period in the control group, and the bodies were confirmed to be positive by an immuno-staining method for α2u globulin antibody.
The nodule macroscopically observed in the epididymis (unilateral) of one animal in the 100 mg/kg group was diagnosed as spermatic granuloma.
Female:
One female of satellite females of the 500 mg/kg group (animal No. 2506)
Inflammation and erosion were observed from the larynx to the upper part of the trachea. In the upper part of trachea, respiratory obstruction with pus was observed. In addition, congestion, edema, and slight hemorrhage were observed in the lung.

No pathological lesions indicative of the influence of administration were observed in any tissues.
Since abnormal respiration was observed in the clinical observation, histopathological examination of the nose was conducted in all cases, but no abnormalities were observed.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

In one animal (animal No. 2308) of the 500 mg/kg group, since its estrus phase disappeared so that a diestrus phase was continued from 4 day of administration to the beginning of mating, this animal was judged to have an abnormal estrous cycle.
No influences of administration were noted in the mean duration of an estrous cycle calculated in each group.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Results of mating
During the mating period for 14 days, all animals mated (the longest case: 5 days).
In addition, a duration necessitated for mating showed no differences among groups, and no influences of administration were noted.
One animal in the 100 mg/kg group (animal No. 2204), and one animal in the 500 mg/kg group (animal No. 2309) did not deliver until equivalent 25 day of pregnancy, and the animals were sacrificed and necropsied on equivalent 26 day of pregnancy. In the results of necropsy, no corpora lutea of pregnancy were seen in the ovaries, and no fetus nor implantation sites were seen in the uterus. Therefore, these 2 animals were judged to be infertile.

Gestation and parturition
No abnormalities were observed in pregnant females (dams) during the gestation period and in parturition. In addition, no influences of administration were noted during the gestation period (a period between mating and delivery).

Implantation
No influences of administration were seen in the number of corpora lutea of pregnancy, implantation sites, and implantation index.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Abnormal respiration was observed in males and females of the 500 mg/kg group.

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No influences due to the test substance administration were observed in the reproductive performances of parental animals of male and female, and in the genesis/development of offspring.

Remarks on result:

other: Generation: P/F1 (migrated information)

Clinical signs:

no effects observed

Description (incidence and severity):

No influences of administration were noted in the number of delivered pups and sex ratio, delivery index, number of live pups and sex ratio on 0 day of lactation, viability index and live birth index on 0 day lactation. In addition, there was no animal with abnormalities in the external observation.
No abnormalities were noted in lactation condition and clinical observation of offspring. No influences of administration were noted.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No influences of administration were noted in the body weights of male and female offspring.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were observed in any pups.

Histopathological findings:

no effects observed

Other effects:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Number of offspring on the delivery day
No influences of administration were noted in the number of delivered pups and sex ratio, delivery index, number of live pups and sex ratio on 0 day of lactation, viability index and live birth index on 0 day lactation, body weights of male and female offspring. In addition, there was no animal with abnormalities in the external observation.

Lactation condition and clinical observation of offspring
No abnormalities were noted in lactation condition and clinical observation of offspring. No influences of administration were noted.

Offspring on 4 day of lactation
No influences of administration were observed on the number of live pups, sex ratio, viability index, and body weights of live male and female pups on 4 day of lactation,.

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Reproductive effects observed:

not specified

Gross findings of rats treated orally with 2,4-dimethylbenzenesulfonic acid in the combined repeated dose and reproductive/developmental toxicity screening test

Dose (mg/kg)	At the end of administration period				At the end of recovery period
	0	20	100	500	0	500
Male
No. of animals examined	7	12	12	7	5	5
Non-remarkable Epididymis, nodule Stomach/intestines, gas	7 0 0	12 0 0	11 1 0	7 0 0	5 0 0	4 0 1
Female
No. of animals examined	12 (5)	12	12	12 (5)	(5)	(4)^1)
Non-remarkable Liver, herniation	12 (5) 0 (0)	12 0	11 1	12 (5) 0 (0)	(5) (0)	(4) (0)

Data represent the number of animals.

Parentheses represent the number of satellite females.

¹⁾1 female dies during administration period. This animal exhibited gaseous distension of the stomach at necropsy.

 

Estrous cycle and reproductive performance of rats treated orally with 2,4-dimethylbenzenesulfonic acid in the combined repeated dose and reproductive/developmental toxicity screening test

Dose (mg/kg)	0	20	100	500
Estrous cycle
No. of animals examined Normal^1) Abnormal^3) Cycle (days)^2)	12 12 0 4.2 ± 0.3	12 12 0 4.1 ± 0.3	12 12 0 4.1 ± 0.2	12 11 1 4.3 ± 0.4
Reproductive performance
No. of mated pairs No. of copulated pairs Copulation index (%) Pairing days until copulation^2) No. of pregnant females Fertility index (%) No. of females with complete parturition Gestation index (%) Gestation length (days)^2) Findings in parturition^1) -No abnormalities detected Findings in nursing^1) -No abnormalities detected	12 12 100.0 2.8 ± 0.9 12 100.0 12 100.0 22.0 ± 0.0   12   12	12 12 100.0 2.6 ± 1.2 12 100.0 12 100.0 22.0 ± 0.0   12   12	12 12 100.0 2.3 ± 1.2 11 91.7 11 100.0 22.1 ± 0.3   11   11	12 12 100.0 2.6 ± 1.2 11 91.7 11 100.0 22.0 ± 0.0   11   11

¹⁾Data represent the number of animals

²⁾Data represent mean ± S.D.

³⁾Continuous diestrus

Copulation index = (No. of copulated pairs / No. of mated pairs) x 100

Fertility index = (No. of pregnant females / No. of copulated pairs) x 100

Gestation index = (No. of female with complete parturition / No. of pregnant females) x 100

 

Summary of implantation and development of pups from dams treated orally with 2,4-dimethlybenzenesulfonic acid in the combined repeated dose and reproductive/developmental toxicity screening test

Dose (mg/kg)	0		20		100		500
No. of corpora lutea	15.8 ± 0.7	(12)	14.6 ± 2.4	(12)	15.4 ± 1.3	(11)	15.3 ± 1.2	(11)
No. of implantations	15.8 ± 0.8	(12)	14.4 ± 2.4	(12)	15.1 ± 1.4	(11)	15.2 ± 1.2	(11)
Implantation index (%)	99.5 ± 1.8	(12)	98.9 ± 2.7	(12)	98.2 ± 3.2	(11)	99.4 ± 1.9	(11)
Postnatal day 0
No. of pups born -No. of males per group -No. of females per group Sex ratio of pups born Delivery index (%) No. of live pups -No. of males per group -No. of females per group Sex ratio of live pups Birth index (%) Live birth index (%)	14.7 ± 1.2 92 84 0.52 ± 0.15 93.1 ± 5.1 14.6 ± 1.2 92 83 0.52 ± 0.15 92.6 ± 5.3 99.4 ± 1.9	(12) (12) (12) (12) (12) (12) (12) (12) (12) (12) (12)	13.4 ± 2.4 77 84 0.48 ± 0.10 93.1 ± 6.3 13.4 ± 2.4 77 84 0.48 ± 0.10 93.1 ± 6.3 100.0 ± 0.0	(12) (12) (12) (12) (12) (12) (12) (12) (12) (12) (12)	13.9 ± 1.4 72 81 0.48 ± 0.14 92.4 ± 6.8 13.9 ± 1.4 72 81 0.48 ± 0.14 92.4 ± 6.8 100.0 ± 0.0	(11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11)	14.2 ± 1.1 87 69 0.56 ± 0.12 93.7 ± 7.0 14.2 ± 1.1 87 69 0.56 ± 0.12 93.7 ± 7.0 100.0 ± 0.0	(11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11)
Pup weight (g)
Male Female	6.9 ± 0.4 6.4 ± 0.4	(12) (12)	6.7 ± 0.4 6.3 ± 0.4	(12) (12)	6.8 ± 0.3 6.4 ± 0.3	(11) (11)	6.9 ± 0.7 6.4 ± 0.7	(11) (11)
Postnatal day 4
No. of live pups -No. males per group -No. females per group Sex ratio of live pups Viability index (%)	14.6 ± 1.2 92 83 0.52 ± 0.15 100.0 ± 0.0	(12) (12) (12) (12) (12)	13.4 ± 2.4 77 84 0.48 ± 0.10 100.0 ± 0.0	(12) (12) (12) (12) (12)	13.5 ± 1.8 69 80 0.47 ± 0.13 97.2 ± 5.4	(11) (11) (11) (11) (11)	14.2 ± 1.1 87 69 0.56 ± 0.12 100.0 ± 0.0	(11) (11) (11) (11) (11)
Pups weight (g)
Male Female	10.8 ± 0.9 10.2 ± 0.8	(12) (12)	10.9 ± 0.6 10.3 ± 0.7	(12) (12)	10.8 ± 0.7 10.5 ± 0.8	(11) (11)	10.8 ± 1.0 10.1 ± 1.0	(11) (11)

Data represent mean ± S.D.

Parentheses represent the number of litters examined.

Implantation index = (No. of implantations / No. of corpora lutea) x 100.

Sex ratio of pups born = No. of male pups born / No. of male and female pups born.

Delivery index = (No. of pups born / No. of implantations) x 100.

Sex ratio on day 0 = No. of male live pups on day 0 / No. of live pups on day 0.

Birth index = (No. of live pups of day 0 / No. of implantations) x 100.

Live birth index = (No. of live pups on day 0 / No. of pups born) x 100.

Viability index = (No. of live pups on day 4 / No. of live pups on day 0) x 100.

Sex ratio on day 4 = No. of male live pups on day 4 / No. of live pups on day 4.

 

External abnormalities, general clinical observations and gross findings in necropsy of pups from dams treated orally with 2,4-dimethylbenzenesulfonic acid in the combined repeated dose and reproductive/developmental toxicity screening test.

Dose (mg/kg)	0	20	100	500
External abnormality
No. of pups examined Findings -No abnormalities detected	175 (12)   175 (12)	161 (12)   161 (12)	153 (11)   153 (11)	156 (11)   156 (11)
General clinical observation
No. of pups examined Findings^1) -No abnormalities detected -Death -Cannibalized	175 (12)   175 (12) 0 (0) 0 (0)	161 (12)   161 (12) 0 (0) 0 (0)	153 (11)   149 (11) 2 (2) 2 (2)	156 (11)   156 (11) 0 (0) 0 (0)
Gross finding in necropsy
No. of pups examined^2) Findings -No abnormalities detected	176 (12)   176 (12)	161 (12)   161 (12)	151 (11)   151 (11)	156 (11)   156 (11)

Parentheses represent the number of litters.

¹⁾Except the external abnormalities.

²⁾All necropsied pups including stillborn.

Conclusions:

NOAEL in reproduction and development toxicity is concluded to be 500 mg/kg/day.

Executive summary:

This study was conducted in order to assess the repeated dose toxicity and reproductive/ developmental toxicity of 2,4-dimethylbenzenesulfonic acid, in accordance with OECD Guideline 422.

In this study, 2,4-dimethylbenzenesulfonic acid was administered to Crl:CD (SD) male and female rats (12 animals/group, 10 weeks old) at the doses of 0 (vehicle control, water for injection), 20, 100, and 500 mg/kg. Daily gavage administration was conducted for 42 days in males, and for 41 to 46 days until 4 day of lactation in females. The influences caused by the repeated administration to males and females, and influences on the reproduction performance of parental animals, and the genesis/development of offspring until 4 day of lactation were assessed. In addition, for the control group and 500 mg/kg group, 5 out of 12 male animals used for mating were assigned for a recovery group. In females, 10 animals were used as a satellite group, which were not used in the reproduction study, and 5 out of these satellite 10 females were assigned for a recovery group. 2 weeks of recovery period after the end of repeated 42 day of administration was set.

 

In the clinical observation during the administration period, abnormal respiration (abnormal respiratory sound, irregular respiration, open mouth respiration) were observed in the 500 mg/kg groups of both sexes. One male in the 500 mg/kg group showed deep respiration from the middle of the recovery period. In females of the 500 mg/kg group, contamination with urine around the external reproductive organ was observed during the administration period. The suppression of body weight increase was observed in male of the 500 mg/kg group. on the other hand, no influences related to administration to the body weight were noted in female animals. One satellite female of the 500 mg/kg group, died suddenly during the administration period. The cause of the death was estimated to be suffocation caused by the obstruction of respiratory tract induced by the inflammation of the upper part of trachea. However, since inflammation of upper part of the trachea was not observed in the other animals, this death was judged to be unrelated to the toxicity of 2,4-dimethylbenzenesulfonic acid.

In the results of hematological examination, MCH was lowered in male of 500 mg/kg group. In the blood biochemistry examination of the 500 mg/kg group, males showed decrease in the levels of total protein and albumin, those females which were pregnant and delivered showed the decrease in BUN, creatinine, and inorganic phosphate. The females of the satellite group showed the decrease in the level of total protein, and the elevation of AST level. In urinalysis, glucose was positive in one female of the satellite 500 mg/kg group. However, all of these changes showed reversibility.

For the organ weight of males and female which were pregnant and delivered, no influences related to administration were noted. In females of the satellite 500 mg/kg group, absolute weights and relative weights of the liver and kidneys was increased at the end of the recovery period. In the histopathological examination, no findings related to administration were noted in both sexes.

No influences related to administration to an estrous cycle, mating, gestation, parturition, and genesis/development of offspring were observed.

 

By repeated oral administration of 2,4-dimethylbenzenesulfonic acid in this study, abnormal respiration was observed in males and females of the 500 mg/kg group, but no influences were noted in the 100 mg/kg group, and therefore, a no observed adverse effect level (NOAEL) is concluded to be 100 mg/kg/day for both sexes. In addition, since no influences due to the test substance administration were observed in the reproductive performances of parental animals of male and female, and in the genesis/ development of offspring, a NOAEL in reproduction and development toxicity is concluded to be 500 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

This study was conducted in order to assess the repeated dose toxicity and reproductive/ developmental toxicity of 2,4-dimethylbenzenesulfonic acid, in accordance with OECD Guideline 422.

In this study, 2,4-dimethylbenzenesulfonic acid was administered to Crl:CD (SD) male and female rats (12 animals/group, 10 weeks old) at the doses of 0 (vehicle control, water for injection), 20, 100, and 500 mg/kg. Daily gavage administration was conducted for 42 days in males, and for 41 to 46 days until 4 day of lactation in females. The influences caused by the repeated administration to males and females, and influences on the reproduction performance of parental animals, and the genesis/development of offspring until 4 day of lactation were assessed. In addition, for the control group and 500 mg/kg group, 5 out of 12 male animals used for mating were assigned for a recovery group. In females, 10 animals were used as a satellite group, which were not used in the reproduction study, and 5 out of these satellite 10 females were assigned for a recovery group. 2 weeks of recovery period after the end of repeated 42 day of administration was set.

 

In the clinical observation during the administration period, abnormal respiration (abnormal respiratory sound, irregular respiration, open mouth respiration) were observed in the 500 mg/kg groups of both sexes. One male in the 500 mg/kg group showed deep respiration from the middle of the recovery period. In females of the 500 mg/kg group, contamination with urine around the external reproductive organ was observed during the administration period. The suppression of body weight increase was observed in male of the 500 mg/kg group. on the other hand, no influences related to administration to the body weight were noted in female animals. One satellite female of the 500 mg/kg group, died suddenly during the administration period. The cause of the death was estimated to be suffocation caused by the obstruction of respiratory tract induced by the inflammation of the upper part of trachea. However, since inflammation of upper part of the trachea was not observed in the other animals, this death was judged to be unrelated to the toxicity of 2,4-dimethylbenzenesulfonic acid.

In the results of hematological examination, MCH was lowered in male of 500 mg/kg group. In the blood biochemistry examination of the 500 mg/kg group, males showed decrease in the levels of total protein and albumin, those females which were pregnant and delivered showed the decrease in BUN, creatinine, and inorganic phosphate. The females of the satellite group showed the decrease in the level of total protein, and the elevation of AST level. In urinalysis, glucose was positive in one female of the satellite 500 mg/kg group. However, all of these changes showed reversibility.

For the organ weight of males and female which were pregnant and delivered, no influences related to administration were noted. In females of the satellite 500 mg/kg group, absolute weights and relative weights of the liver and kidneys was increased at the end of the recovery period. In the histopathological examination, no findings related to administration were noted in both sexes.

No influences related to administration to an estrous cycle, mating, gestation, parturition, and genesis/development of offspring were observed.

 

By repeated oral administration of 2,4-dimethylbenzenesulfonic acid in this study, abnormal respiration was observed in males and females of the 500 mg/kg group, but no influences were noted in the 100 mg/kg group, and therefore, a no observed adverse effect level (NOAEL) is concluded to be 100 mg/kg/day for both sexes. In addition, since no influences due to the test substance administration were observed in the reproductive performances of parental animals of male and female, and in the genesis/ development of offspring, a NOAEL in reproduction and development toxicity is concluded to be 500 mg/kg/day.

Justification for selection of Effect on fertility via oral route:

The data display the most conservative results are are therefore considered most appropriate to hazard assessment

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

The data available by read across to 2,4-dimethylbenzenesulfonic acid demonstrate toxicity to parental animals but no inhibition to the reproduction and no apparent toxicity to offspring following subacute exposure.