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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-methyl-5-nitrobenzenesulfonic acid
- Molecular formula (if other than submission substance): 217.20
- Physical state: pale yellow granules
- Analytical purity: 79.6 %

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 309-335 g; females: 202-232 g
- Housing:individually; 2 cubicles of a metal wire-mesh cage
- Diet (e.g. ad libitum): ad libitum (NMF pellet, Oriental Yeast Co.,Ltd.)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24.5
- Humidity (%): 40.5 - 71.5
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The preparation was made once or more a week, stored in a cold, dark place.

Details on mating procedure:
1 each of male and female (a total of 2 rats) were placed in the cage from evening to next morning every day.
The cohabitation period was up to 14 days, until copulation was confirmed. Copulation was checked based on the presence or absence of vaginal plug or sperms in vaginal smear every morning; and the day of discovering such evidence was defined as day 0 of gestation.
Duration of treatment / exposure:
male: until days 49 or 50 (inculding a 14-day premaiting and maiting period)
female: until days 41-48 (including a 14-day pre-maiting and gestation period until day 3 of lactation)
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 175, 350, 700 mg/kg
Basis:

No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
General medical conditions were observed every day throughout the test period in both males and females.

To observe parturition, all females, in which copulation was confirmed, underwent spontaneous delivery. The completion of parturition was confirmed twice a day (once in the morning and once in the afternoon) during days 21-24 of gestation, and once in the morning on day 25 of gestation. When parturition was completed by 10:30 a.m., the current day was defined as day 0 of lactation. In contrast, when parturition was completed after 10:30 a.m., the next day was defined as day 0 of lactation. If parturition was not confirmed even on day 25 of gestation, the animal was subjected to autopsy to measure success and failure of gestation.

To observe a lactation state, mother animals, in which the completion of parturition was confirmed, were made nurse live pups; and the observation was carried out every day until day 4 of lactation. Based on these observation results, the gestation period [day 0 of lactation (the day when parturition was confirmed) - day 0 of gestation], birth rate [(number of females delivering live newborns/number of pregnant animals) x 100], implantation rate [(number of implantation sites/number of corpora lutea) x 100], delivery rate [(total number of live newborns/number of implantation sites) x 100] were calculated.

BODY WEIGHT: Yes
- Time schedule for examinations:
measured on days 1 (starting day of administration), 4, 8, 11, 15, 22, 29, 36, 43, 49 and 50 or 51 (day of autopsy) of administration in males. In females, body weights were measured: on days 1, 4, 8, 11 and 15 of administration during the pre-mating period; on day 22 of administration during the mating period; on days 0, 7, 14 and 21 of gestation during the gestation period; and days 0 and 4 (day of autopsy) of lactation during the lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
In males, food consumption was estimated on the same days as body weight measurement, except for the mating period and autopsy day.
In females, food consumption was estimated: on the same days as body weight measurement during the pre-mating period; on days 1, 7, 14 and 21 of gestation during the gestation period; and on days 1 and 4 of lactation during the lactation period.

HAEMATOLOGY/CLINICAL CHEMISTRY: Yes
males animals: blood samples from the abdominal aorta
Some of these blood samples were used (after adding EDTA-2K thereto) to measure/calculate the red blood cell count, hemoglobin content, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count and white blood cell count (electric resistance change detecting method and cyanmethemoglobin method) by using a Coulter T890 automated hematology analyzer (for 8 items) [Nikkaki Bios Co., Ltd.], and methemoglobin content (Van Assendelft method) by using a UVIDEC-66 spectrophotometer [JASCO Medical Instruments], respectively.
Using blood plasma samples obtained by adding 3.8 %- sodium citrate to some of the collected blood samples and by conducting centrifugation (3000 rpm, 10 min.), the prothrombin time, activated partial thromboplastin time and fibrinogen content (clot method and thromboplastin method) were measured by means of an ACL100 automated blood coagulation analyzer [Instrumentation Laboratory]. Furthermore, the collected blood samples were used to obtain the white blood cell differential count by observing May-Giemsa-stained smears, and the reticulocyte percentage by observing supravitally stained samples according to the Brecher method.
GOT, GPT, LDH (UV-rate method) were measured ba unsing boold plasma samples.
Oestrous cyclicity (parental animals):
Vaginal smears were collected for microscopy every day throughout the period from the day after administration until confirmation of mating, in order to obtain the estrous cycle as the frequency of expressing an estrous state and the interval of days between estrous periods.
Postmortem examinations (parental animals):
700 mg/kg: 4 males and 3 females exhibited stridor on and after day 8 of administraion
Additionally, soft feces with partial black changes, abdominal distension, soiled perineal region, unkempt fur or underdeveloped nipples were seen insome animals

CLINICAL SIGNS AND MORTALITY
In the group of 700 mg/kg, 1 female died during the mating period, while 1 female was sacrificed in extremis during the gestation period. In the death case, the animal exhibited sequentially stridor, soft feces with partial black changes, paleness of pinna, soiled perineal region and emaciation on and after day 19 of administration, and died on day 27 of administration. At autopsy, emaciation, soiled perineal region, atrophies of the thymus, spleen and uterus as well as swelling of the lungs were seen. In histopathological examination, mild peribronchiolar inflammatory cell infiltrate in the lungs, severe atrophy in the thymus, moderately-severe atrophy in the spleen, very mild brown pigmentation in the splenic red pulp, and mild atrophy in the uterus were observed.

In the case of sacrifice in extremis, the animal showed stridor on day 12 of administration, and additionally stridor, abdominal distension, piloerection, emaciation, decreased spontaneous movement, hypothermia and oligopnea on days 3-5 of gestation; and thus the female was sacrificed in extremis on day 5 of gestation. At autopsy, atrophy in the spleen, swelling of the adrenal glands, gas retention and dilation in the stomach and enteric canal, partial thickening of the glandular stomach, emaciation, piloerection and abdominal distension were found. In histopathological examination, severe mucosal necrosis of the glandular stomach, mild hemorrhage and moderately-severe edema of the submucous tissue in the glandular stomach, moderately-severe inflammatory cell infiltrate in the glandular stomach, very mild erosion of the limiting ridge, very mild mucosal atrophies in the duodenum, jejunum and ileum, moderately-severe atrophy in the spleen, and very mild brown pigmentation in the splenic red pulp were observed.

ORGAN WEIGHTS
In the 700 mg/kg group, males showed a significantly low value for absolute weight of the liver, as well as significantly high values for relative weights of the kidneys, testes and epididymides; while females exhibited significantly high values for relative weights of the heart and kidneys. However, there were changes only in absolute or relative weights, and no histological abnormalities were seen. Accordingly, it was considered that such changes were ascribable to low body weights.

AUTOPSY FINDINGS
Thickening of the limiting ridge in the stomach was seen in 1 male of the 350 mg/kg group and in 9 males and 2 females of the 700 mg/kg group. Two males separately exhibited dark-red spots or erosion in the glandular stomach in the 700 mg/kg group. Partially recessed areas in the glandular stomach were found in 1 male of the 700 mg/kg group. In addition, partially recessed areas in the kidney (unilateral) and caudal tuber in the epididymis (unilateral) were observed in males of the control group. Dark-red spots in the lungs were found in females of the 175 mg/kg group. White spots in the glandular stomach were observed in females of the 350 mg/kg group. In the 700 mg/kg group, males exhibited partially browning in the lungs and atrophy in the thymus, while 2 females separately showed underdeveloped nipples and mammary glands or dark-red spots in the lungs; however, in view of the nature of these lesions and their manifestation conditions, it was considered that they had no relationship with the administration of the test substance.

HISTOPATHOLOG
Changes caused by the administration of the test substance were found in the stomach in males and females of the 350 and 700 mg/kg groups. More specifically, very mild or mild mucosal hyperplasia in the limiting ridge was seen in 9 males and 3 females of the 350 mg/kg group and in 11 males and 7 females of the 700 mg/kg group; very mild or mild mucosal atrophy in the cardiac region was seen in 9 males and 3 females of the 350 mg/kg group and in all males and females of the 700 mg/kg group; very mild to moderately-severe erosion (including healed erosion) in the glandular stomach was found in 6 males and 5 females of the 700 mg/kg group; and very mild or mild superficial hemorrhage in the glandular stomach was found in 5 males and 1 female of the 700 mg/kg group. In these findings, the manifestation frequencies of mucosal hyperplasia in the limiting ridge and mucosal atrophy in the cardiac region were significantly high in males of the 350 mg/kg group and in males and females of the 700 mg/kg group; and the level was higher in the 700 mg/kg group than that in the 350 mg/kg group. The manifestation frequency of erosion (including healed erosion) in the glandular stomach was also significantly high in males of the 700 mg/kg group. In addition, mild focal hyperplasia in the forestomach mucosa was seen in 1 female of the 350 mg/kg; while myocardial degeneration/fibrosis, extramedullary hematopoiesis and microgranuloma in the liver, extramedullary hematopoiesis in the spleen, brown pigmentation in the splenic red pulp, focal fibrosis in the renal cortex, basophilic degeneration of the renal tubules, oxyphilic corpuscles in the tubular epithelium and spermatic granuloma in the epididymides were found in the control or 700 mg/kg group. However, there were no histopathological characteristics, no growing manifestation frequencies and no raised levels along with dose increase; and consequently, it was considered that they all had no relationship with the administration of the test substance. Furthermore, also grossly abnormal sites underwent the histopathological test, and there were no changes indicating a relationship with the administration of the test substance.
Postmortem examinations (offspring):
OBSERVATION
On day 0 of lactation, the numbers of live pups and stillborns were counted to examine their sex and the presence or absence of external anomalies. All live pups were nursed by their mother animals to observe life and death once a day. From these observation results, the sex ratio (male/female), live birth rate [(number of live pups on day 0 of lactation/total number of live newborns) x 100], viability of newborns on day 4 [(number of live pups on day 4 of lactation / number of live pups on day 0 of lactation) x 100] were obtained.

BODY WEIGHT
Body weights were measured on days 0 and 4 of lactation to obtain each average value for male and female on a per-litter basis.

AUTOPSY
On day 4 of lactation, all live pups were subjected to autopsy after death from exsanguination under ether anesthesia, in order to observe the presence or absence of anomalies of internal organs.
Statistics:
Bartlett method, Dunnett`s method, Kruskal-Wallis rank test, Mann-Whiteny U test
The levels of significance were set to 5 and 1 %.

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

BODY WEIGHT AND WEIGHT GAIN
In males of the 700 mg/kg group, retarded body weight gain were found on and after day 4 of administration, and significantly low values were noted in comparison with the control group on and after day 15 of administration.
In females of the 700 mg/kg group, body weight showed a tendency toward a low value on days 8, 11 and 15 of administration during the pre-mating dosing period, and body weight gain exhibited a significantly low value during the same period. In the same group, the tendency toward a low value for body weight further continued throughout the gestation and lactation period; however, no significant differences were found compared to the control group.
In males and females of the 175 and 350 mg/kg groups, body weights and food consumptions showed the same trends as those in the control group throughout the dosing period, and there were no effects of the test substance.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In males of the 700 mg/kg group food consumption showed a tendency toward a low value on days 29 and 36 of administration, and a significantly low value on day 43 of administration.
In females of the 700 mg/kg group, food consumption showed a value equivalent to that in the control group.
In males and females of the 175 and 350 mg/kg groups, body weights and food consumptions showed the same trends as those in the control group throughout the dosing period, and there were no effects of the test substance.

HAEMATOLOGY
In hematological test, significantly low values for mean corpuscular volume and mean corpuscular hemoglobin were found in the 700 mg/kg group.

CLINICAL CHEMISTRY
On myelogram, there were no changes concerning the erythroid cell, myelocytic cell and M/E ratios associated with the administration of the test substance.

In blood biochemical test, a significantly high value for serum iron was seen in the 700 mg/kg group. In addition, significantly low values for total protein and the α1-globulin fraction ratio in the protein fraction were seen in the 700 mg/kg group; however, there were no changes indicating histological abnormalities or hypo-functions in the liver and kidneys.

REPRODUCTIVE FUNCTION
In the administration groups of the test substance, the estrous cycle, i.e. the frequency of expressing an estrous state and the interval of days between estrous periods during the observation period, was almost the same as that in the control group; and there were no significant differences. With regard to the mating results, almost all couples of the administration groups of the test substance copulated within 5 days from the beginning of cohabitation, and the copulation rate was 100 %. Additionally, there were no significant differences in the number of days required until copulation. Moreover, the conception rate in each administration group of the test substance was 90.9-100 %; and there were no significant differences compared to that in the control group.

In all females except for sterile cases, the completion of parturition was confirmed on or before day 23 of gestation; and no dystocia was seen. Additionally, in each administration group of the test substance, the gestation period, number of corpora lutea and implantation sites, implantation rate, birth rate, delivery rate and live birth rate were equivalent to those in the control group; and there were no significant differences.

As for the lactation state, in all cases of each group, there were no abnormalities in lactation behaviors, such as nest-building, pup-gathering and suckling.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
700 mg/kg bw/day
Sex:
male/female

Results: F1 generation

Details on results (F1)

In external examination of newborns, every group showed no anomalies. In each administration group of the test substance, the sex ratio and the number of live pups were almost equivalent to those in the control group; and no significant differences were observed. However, male and female bodyweights on days 0 and 4 of lactation and the viability of newborns on day 4 of lactation showed a tendency toward low values in the 700 mg/kg group. At autopsy on day 4 of lactation, there were no changes caused by the administration of the test substance.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
350 mg/kg bw/day
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Summary of reproductive perfomance of female rats

Dose (mg/kg/day)

0

175

350

700

Estrus cycle (days, Mean±S.D.

4.2 ± 0.5

4.8 ± 2.0

4.3 ± 0.4

4.3 ± 0.4

No. of pairs mated

12

12

12

11

No. of pairs copulated

12

12

12

11

No. of pregnant females

10

12

11

10

Copulation index (%)

100.0

100.0

100.0

100.0

Fertility index (%)

83.3

100.0

91.7

90.9

 

 

Body weight of pups from dams 

Dose (mg/kg/day)

0

175

350

700

Day 0 of lactation

 

 

 

 

No. of dams

10

12

11

9

Male (g)

7.2 ± 0.8

7.2 ± 0.7

7.0 ± 0.5

6.7 ± 0.3

Females (g)

6.9 ± 0.7

6.8 ± 0.6

6.7 ± 0.5

6.3 ± 0.4

Day 4 of lactation

 

 

 

 

No. of dams

10

12

11

9

Male (g)

11.3 ± 2.7

11.1 ± 1.2

10.7 ± 0.8

10.0 ± 2.3

Female (g)

10.8 ± 2.2

10.5 ± 0.8

10.1 ± 0.7

9.5 ± 2.2

  

Delivery and litter data of female rats 

Dose (mg/kg/day)

0

175

350

700

No. pf pregnant females

10

12

11

9

No. of females with live pups

10

12

11

9

Gestation index (%)

100.0

100.0

100.0

100.0

Gestation length (days, Mean±S.D.)

22.7 ± 0.5

22.7 ± 0.5

22.5 ± 0.5

22.3 ± 0.5

No. of corpora lutea (Mean±S.D.)

185 (18.5 ± 2.4)

226 (18.8 ± 1.6)

214 (19.5 ± 2.0)

163 (18.1 ± 2.7)

No. of implantations (Mean±S.D.)

180 (18.0 ± 2.7)

212 (17.7 ± 1.7)

206 (18.7 ± 1.8)

161 (17.9 ± 2.8)

Implantation index (%)

97.1

94.1

96.3

98.8

No. of stillborn (Mean±S.D.)

1 (0.1 ± 0.3)

1 (0.1 ± 0.3)

1(0.1 ± 0.3)

0 (0.0 ± 0.0)

No. of live born (Mean±S.D.)

155 (15.5 ± 4.7)

187 (15.6 ± 2.0)

193 (17.5 ± 1.6)

147 (16.3 ± 2.6)

Live birth index (%)

99.6

99.5

99.5

100.0

Delivery index (%)

85.5

88.8

94.5

91.5

No. of male pups (Mean±S.D.)

78 (7.8 ± 3.0)

91 (7.6 ± 2.7)

94 (8.5 ± 1.3)

69 (7.7 ± 1.9)

No. of female pups (Mean±S.D.)

77 (7.7 ± 4.0)

96 (8.0 ± 2.7)

99 (9.0 ± 1.5)

78 (8.7 ± 2.6)

Sex ration

1.01

0.95

0.95

0.88

No. of live pups (Mean±S.D.)

 

 

 

 

Day 0 of lactation

155 (15.5 ± 4.7)

187 (15.6 ± 2.0)

193 (17.5 ± 1.6)

147 (16.3 ± 2.6)

Day 4 of lactation

153 (15.3 ± 4.5)

184 (15.3 ± 2.3)

187 (17.0 ± 1.5)

128 (14.2 ± 5.6)

Viability index (%)

99.0

98.2

97.0

85.9

 

 

Applicant's summary and conclusion