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Toxicological information

Developmental toxicity / teratogenicity

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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:
other: GLP-Guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: 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
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 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

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

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.
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 (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
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.

With regard to the effects on newborns, also in the 700 mg/kg group, no external anomalies were observed in newborns. And there were no effects of the test substance on the sex ratio, number of live newborns, number of stillborns and live birth rate. However, in the 700 mg/kg group, there were tendencies toward low body weight in males and females and toward low viability on day 4 of lactation. These changes were presumed to be secondary ones, which arose from toxic changes during the gestation and lactation periods of the mother.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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