Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Twenty-five respectively thirty (250 mg/kg group, only) inseminated female Wistar rats each were treated daily orally by gavage with Dichlortoluol (purity 99.1 %) in polyethylene glycol 400 from day 6 to day 20 p.c. in the following doses: 0, 250, 500, and 750 mg/kg body weight (bw)/day, respectively. The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Dichloromethylbenzene
EC Number:
249-854-8
EC Name:
Dichloromethylbenzene
Cas Number:
29797-40-8
Molecular formula:
C7H6Cl2
IUPAC Name:
(dichloromethyl)benzene
Test material form:
other: liquid
Details on test material:
content: ca. 99.1%

The test item was administered by gavage in polyethylene glycol 400. The administration volume was 5 mL/kg body weight. The formulations were prepared as needed taking into account the analytically determined stability.

The test substance was administered as a solution in the vehicle. The formulations were stored at room temperature. Before the start of treatment, the suitability of the formulation was confirmed by the analysis of concentration and stability of dosage forms prepared in the same way as it were done in the study (2 and 200 mg/mL, covering the concentration range used). Analyses were carried out before the start of the study under 2012/0029/07. Content checks of the active ingredient in samples with concentrations of 50, 100, and 150 mg/mL were carried out twice during the study on February 04, 2013, and March 04, 2013.

Stability was confirmed in the investigation on the stability of the active ingredient after 8 days of storage with concentrations of 2 and 200 mg/mL before the start of the study.
The results of the content checks in samples with concentrations of 50, 100, and 150 mg/mL during the study showed no meaningful deviation of the active ingredient content from the nominal value.

Test animals

Species:
rat
Strain:
Wistar

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
The male animals were used for mating only and were not treated. After insemination was ascertained, 100 females were allocated to four experimental groups according to a computer generated randomization plan (random number generator). Due to an incidentally lower fertility rate in the 250 mg/kg group, five additional inseminated females were allocated to this group.

The females were treated daily between 06.00 and 12.00 CET from days 6 to 20 of gestation. The females received the administration formulations orally by gavage.
The females in all experimental groups received a uniform administration volume of 5 mL/kg body weight/day. The dose volume was adjusted to the current body weight which was determined before each administration daily from days 6 to 20 p.c. The females of the control group received vehicle, only.
The following doses were administered once daily corresponding to the concurrent body weight:
Table 4 2: Dose Schedule
Group No. Dose (mg/kg) Concentration (mg/mL) Number of Animals
Group 1 (Control) 0 0 25
Group 2 (Low dose) 250 50 30
Group 3 (Medium dose) 500 100 25
Group 4 (High dose) 750 150 25

The administration volumes, corresponding to the daily body weights, were recorded on-line and are filed together with the study raw data but are not reported.

The dose levels were selected based on the results of a previous pilot prenatal developmental toxicity study in rats with Dichlortoluol, which revealed only slight maternal toxicity at the 500 mg/kg level, whereas 1000 mg/kg led to death or severe maternal toxicity so that 750 mg/kg was chosen as the high dose level.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability was confirmed in the investigation on the stability of the active ingredient after 8 days of storage with concentrations of 2 and 200 mg/mL before the start of the study.
The results of the content checks in samples with concentrations of 50, 100, and 150 mg/mL during the study showed no meaningful deviation of the active ingredient content from the nominal value.
Details on mating procedure:
The animals were mated by placing two females overnight into a Type IIIh cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.
Duration of treatment / exposure:
Once daily, from day 6 p.c. to day 20 p.c.
Frequency of treatment:
Once daily
Duration of test:
The females were sacrificed on day 21 p.c.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
250 mg/kg bw/day
Remarks:
Dichlortoluol
Dose / conc.:
500 mg/kg bw/day
Remarks:
Dichlortoluol
Dose / conc.:
750 mg/kg bw/day
Remarks:
Dichlortoluol
No. of animals per sex per dose:
Twenty-five respectively thirty (250 mg/kg group, only) inseminated female Wistar rats.
Control animals:
yes, concurrent vehicle
Details on study design:
This study was conducted in compliance with the following guidelines:
OECD guidelines: Guidelines for Testing of Chemicals, Section 4: Health Effects, No. 414 “Prenatal Developmental Toxicity Study“, adopted January 22, 2001.
US-EPA: Health Effects Test Guidelines OPPTS 870.3700: “Prenatal Developmental Toxicity Study“, EPA 712-C-98-207, dated August 1998.
Japanese MAFF guidelines: Guideline on the Compiling of Test Results on Toxicity “Teratology Study”, 12-Nousan No. 8147 of November 24, 2000, amended June 26, 2001.
EEC guidelines: Commission Directive 2004/73/EEC, Official Journal of the European Communities L 152, dated April 29, 2004.

Examinations

Maternal examinations:
Clinical Examinations
The females were inspected from days 0 to 21 p.c. twice daily (once daily only on weekends, on public holidays, and on day 21 p.c.), and all findings were recorded. Attention was paid to disturbances in the rats' general condition (appearance, behavior) and alterations concerning their excretory products.

Body Weights
The body weight of the females was determined on day 0 p.c. and daily from day 6 to day 21 p.c. Corrected body weight gain was determined by subtracting the uterus weight on day 21 p.c. from the body weight gain from days 0 to 21 p.c.

Food Intake, Water Intake
The food intake of the animals was determined from the difference in weight between the food offered and the food not consumed for the following days of gestation: Days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, and 18 -21.
Water intake was assessed daily by visual estimation of the quantities left over and reported together with clinical findings.
Ovaries and uterine content:
Necropsy
The females were subjected to gross pathological evaluation at cesarean section on day 21 p.c. Necropsy was performed without knowledge of treatment groups.

Cesarean sections were performed on day 21 p.c. without knowledge of treatment groups. The females were sacrificed by cardiotomy under deep carbon dioxide anesthesia. The fetuses were sacrificed by carbon dioxide asphyxia. The following parameters were determined and assessed at cesarean section and during the subsequent fetal evaluation without knowledge of treatment groups:
• number of corpora lutea
• number of implantations (in females without visible implantation sites after staining of the uterus with a solution of 10 % ammonium sulfide (1))
• uterine weights
• individual weight and appearance of the placentas
• number of early resorptions (only implantation site visible), late resorptions (fetal or placental remnant visible), and dead fetuses (fetuses without signs of life, but without maceration)
Fetal examinations:
Cesarean sections were performed on day 21 p.c. without knowledge of treatment groups. The females were sacrificed by cardiotomy under deep carbon dioxide anesthesia. The fetuses were sacrificed by carbon dioxide asphyxia. The following parameters were determined and assessed at cesarean section and during the subsequent fetal evaluation without knowledge of treatment groups:

• number of live fetuses
• sex of live fetuses
• individual weights of live fetuses
• external malformations and other findings deviating from normal
• visceral malformations and other findings deviating from normal [evaluation of about half of the fetuses by scalpel and/or razor blade sectioning according to the modified WILSON technique (2) (processing and evaluation after cesarean section without knowledge of treatment group)].
• findings in abdominal, pelvic, and thoracic organs as well as skeletal and cartilage findings by the modified DAWSON technique (3) with the addition of cartilage staining [method described by INOUYE, modified (4)]: evisceration, cartilage staining with alcian blue GX, clearing of the fetuses with diluted potassium hydroxide solution, staining of the skeletal system with alizarin red S, and evaluation of the skeletal system including cartilaginous findings (processing and evaluation after cesarean section without knowledge of treatment group). Every other fetus within a litter was prepared for either skeletal or visceral evaluation with generally the first fetus of each litter assigned to skeletal evaluation. The visceral findings observed during the evisceration of fetuses assigned for skeletal evaluation are listed together with the external findings in the Annex of this report.
The term of combined finding of the skeleton or viscera was used in case a comprehensive description was suitable or necessary. The term does not include a classification of the finding (malformation or deviation).
The following Table 4 4 gives an overview on the number of fetuses examined by standardized methods ((5) and (6)):
Table 4 4: Number of Fetuses Examined by Standardized Methods
Dose
[mg/kg bw/day] Total number
of fetuses Fetuses investigated
according to
mod. WILSON mod. DAWSON/ INOUYE
0 295 143 152
250 337 163 174
500 305 147 158
750 304 145 159

Statistics:
Females without implantation sites were not taken into account for calculation of mean values.
The mean values in the tables calculated by computer are the rounded results of the calculations with unrounded raw data values.
In case of skeletal localizations with mechanical damage, these were excluded from the calculation of percentages of affected localizations. The tables of individual skeletal findings enumerate those findings, for which the affected localizations were excluded from calculation.
Differences between the control and Dichlortoluol-treated groups were considered to be significant when p < 0.05. Significant differences from the control group are indicated with * for p < 0.05 and ** for p < 0.01.
Statistical evaluation was performed using the following methods:
a. Analysis of Variance (ANOVA); in case of significance Dunnett's test for
feed intakes
body weights, body weight gains, and corrected body weight gains
uterine weights
number of corpora lutea per femalenumber of implantations per female
number of live fetuses per female and as percentage of implantations per female
placental weights per female
fetal weights per female

b. 2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for
fertility rate
gestation rate
number of implantations per group
number of preimplantation losses per group
number of postimplantation losses, early resorptions, late resorptions or dead fetuses per group
number of live fetuses per group as percentage of implantations per group
number of male or female fetuses or fetuses with indeterminable sex per group
number of placentas with findings or litters with placental findings per group
number of fetuses or litters with external, visceral or skeletal findings, with malformations or with external or visceral deviations per group
Skeletal localizations with mechanical damage in single fetuses were excluded from the calculation of percentages of affected localizations. The tables of
Historical control data:
available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
One female (no. 55) of the 750 mg/kg group was sacrificed in moribund condition on day 9 p.c. after it had shown respiratory sounds, piloerection, reddish substance at mouth and nose, severe body weight loss, and distinctly reduced feed intake. Necropsy of this female revealed small and large intestines (including cecum) with gaseous contents. One further female (no. 69) of this dose group was found dead on day 7 p.c. Necropsy of this female
showed a thoracic cavity filled with clear fluid and a perforated esophagus so that death of this female was caused by misapplication and was thus not considered as a treatment related effect.
Another female of the 750 mg/kg group showed piloerection and respiratory sounds. No further treatment related clinical findings occurred at dose levels up to 750 mg/kg bw/day.
Thus, one female of the 750 mg/kg group was sacrificed in moribund condition after it had shown signs of distinct maternal toxicity, and another female of this dose group was found dead most probably due to a misapplication. Except one further case of piloerection at this dose level, no further treatment related clinical findings occurred in the remaining females a dose levels up to 750 mg/kg bw/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (no. 55) of the 750 mg/kg group was sacrificed in moribund condition on day 9 p.c. after it had shown respiratory sounds, piloerection, reddish substance at mouth and nose, severe body weight loss, and distinctly reduced feed intake. Necropsy of this female revealed small and large intestines (including cecum) with gaseous contents. One further female (no. 69) of this dose group was found dead on day 7 p.c. Necropsy of this female
showed a thoracic cavity filled with clear fluid and a perforated esophagus so that death of this female was caused by misapplication and was thus not considered as a treatment related effect.
Another female of the 750 mg/kg group showed piloerection and respiratory sounds. No further treatment related clinical findings occurred at dose levels up to 750 mg/kg bw/day.
Thus, one female of the 750 mg/kg group was sacrificed in moribund condition after it had shown signs of distinct maternal toxicity, and another female of this dose group was found dead most probably due to a misapplication. Except one further case of piloerection at this dose level, no further treatment related clinical findings occurred in the remaining females a dose levels up to 750 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight loss occurred at the 750 mg/kg group between days 6-8 p.c., and absolute and corrected body weight gains were also decreased by treatment at dose levels of 500 mg/kg bw/day and above and resulted in statistically significantly reduced final body weights.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was decreased between days 6-12 p.c. at dose levels of 500 mg/kg and above, whereas water intake and fecal and urinary excretions were unaffected by treatment at dose levels up to 750 mg/kg bw/day.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Food intake was decreased between days 6-12 p.c. at dose levels of 500 mg/kg and above, whereas water intake and fecal and urinary excretions were unaffected by treatment at dose levels up to 750 mg/kg bw/day.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One female (no. 55) of the 750 mg/kg group, which was sacrificed in moribund condition on day 9 p.c., revealed small and large intestines (including cecum) with gaseous contents. One further female (no. 69) of this dose group, which was found dead on day 7 p.c., showed a thoracic cavity filled with clear fluid and a perforated esophagus so that death of this female was caused by misapplication and was thus not considered as a treatment related effect.
Furthermore, dilation of renal pelvis occurred at all dose levels, for which a treatment related effect is not assumed due to a lacking dose dependency, since this finding also occurred in the current control group, and because this finding is known as a spontaneous finding in the rat strain used.
Thus, one female of the 750 mg/kg group, which was sacrificed in moribund condition, revealed small and large intestines (including cecum) with gaseous contents at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, and fetal sex distribution were unaffected by treatment at dose levels up to 750 mg/kg bw/day,
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, and fetal sex distribution were unaffected by treatment at dose levels up to 750 mg/kg bw/day,
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, and fetal sex distribution were unaffected by treatment at dose levels up to 750 mg/kg bw/day,
Early or late resorptions:
no effects observed
Description (incidence and severity):
The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, and fetal sex distribution were unaffected by treatment at dose levels up to 750 mg/kg bw/day,
Dead fetuses:
no effects observed
Description (incidence and severity):
The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, and fetal sex distribution were unaffected by treatment at dose levels up to 750 mg/kg bw/day,
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, and fetal sex distribution were unaffected by treatment at dose levels up to 750 mg/kg bw/day,
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: decreased food intake and body weight loss

Details on maternal toxic effects:
Appearance, Behavior, and Mortality
One female (no. 55) of the 750 mg/kg group was sacrificed in moribund condition on day 9 p.c. after it had shown respiratory sounds, piloerection, reddish substance at mouth and nose, severe body weight loss, and distinctly reduced feed intake. Necropsy of this female revealed small and large intestines (including cecum) with gaseous contents. One further female (no. 69) of this dose group was found dead on day 7 p.c. Necropsy of this female showed a thoracic cavity filled with clear fluid and a perforated esophagus so that death of this female was caused by misapplication and was thus not considered as a treatment related effect.
Another female of the 750 mg/kg group showed piloerection. No further treatment related clinical findings occurred at dose levels up to 750 mg/kg bw/day.
Thus, one female of the 750 mg/kg group was sacrificed in moribund condition after it had shown signs of distinct maternal toxicity, and another female of this dose group was found dead most probably due to a misapplication. Except one further case of piloerection at this dose level, no further treatment related clinical findings occurred in the remaining females at dose levels up to 750 mg/kg bw/day.

Body Weights
Table 5 1 gives an overview of the mean body weight gain of the females with viable fetuses at cesarean section.
Table 5 1: Mean Body Weight Gain
Dose (mg/kg b.w./day) 0 250 500 750
absolute body weight gain (g) days 6 - 20 p.c.
103.0 98.5 87.8** 82.1**
absolute body weight gain (g) days 0 - 21 p.c.
144.9 138.5 125.0** 118.4**
corrected body weight gain (g) days 0 - 21 p.c.
46.4 43.5 33.5** 28.6**
Statistically significant difference to control ** = p < 0.01

Mean body weight loss occurred at the 750 mg/kg group between days 6-8 p.c., and absolute and corrected body weight gains were also decreased by treatment at dose levels of 500 mg/kg bw/day and above and resulted in statistically significantly reduced final body weights.

Food and Water Intake, Excretory Products
Table 5 2 gives an overview on the food intakes of females with viable fetuses at cesarean section.
Table 5 2: Mean Food Intake
Dose (mg/kg b.w./day) 0 250 500 750
mean food intakes (g/animal/day)
days 0 - 3 p.c. 22.9 23.4 23.1 21.9
days 3 - 6 p.c. 25.8 23.8 23.7 23.6
days 6 - 9 p.c. 25.5 24.1 19.7** 16.7**
days 9 - 12 p.c. 25.3 24.2 22.1** 20.7**
days 12 - 15 p.c. 25.6 24.0 23.2 22.7
days 15 - 18 p.c. 26.3 25.8 25.8 27.0
days 18 - 21 p.c. 27.7 27.4 25.6 26.3
Statistically significant difference to control ** = p < 0.01

Food intake was decreased between days 6-12 p.c. at dose levels of 500 mg/kg and above, whereas water intake and fecal and urinary excretions were unaffected by treatment at dose levels up to 750 mg/kg bw/day.

Necropsy
One female (no. 55) of the 750 mg/kg group, which was sacrificed in moribund condition on day 9 p.c., revealed small and large intestines (including cecum) with gaseous contents. One further female (no. 69) of this dose group, which was found dead on day 7 p.c., showed a thoracic cavity filled with clear fluid and a perforated esophagus so that death of this female was caused by misapplication and was thus not considered as a treatment related effect.
Furthermore, dilation of renal pelvis occurred at all dose levels, for which a treatment related effect is not assumed due to a lacking dose dependency, since this finding also occurred in the current control group, and because this finding is known as a spontaneous finding in the rat strain used.
Thus, one female of the 750 mg/kg group, which was sacrificed in moribund condition, revealed small and large intestines (including cecum) with gaseous contents at necropsy.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The fetal weights were marginally to slightly decreased at dose levels of 250 mg/kg bw/day and above.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Postimplantation loss and correspondingly the number of fetuses were unaffected by treatment at dose levels up to 750 mg/kg.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Fetal sex distribution was unaffected by treatment at dose levels up to 750 mg/kg.
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
A treatment related effect on external and visceral deviations was not evident at dose levels up to 750 mg/kg.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) is assumed for retarded ossification of several localizations (digits, metacarpals, toes, metatarsals, sternebrae, cervical vertebral bodies, caudal vertebral bodies) at the 750 mg/kg level, and it cannot totally be excluded for retarded ossification of several localizations at the 500 mg/kg level and for borderline effects at the 250 mg/kg level in relation to dose-dependently though only marginally to slightly decreased fetal weights at all dose levels. Furthermore, a treatment related effect cannot be excluded for increased incidences of 14th ribs at the 750 mg/kg and 500 mg/kg levels.
Visceral malformations:
no effects observed
Description (incidence and severity):
A treatment related effect on external and visceral deviations was not evident at dose levels up to 750 mg/kg.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: retarded ossification

Details on embryotoxic / teratogenic effects:
General Reproduction Data
Dose (mg/kg b.w./day) 0 250 500 750
inseminated females 25 30 25 25
inseminated females evaluated 25 30 25 23#
females with implantations 21 24 22 21
in % of those inseminated 84.0 80.0 88.0 91.3
mean values per female with implantation sites
corpora lutea 14.4 14.6 15.0 15.2
preimplantation loss 0.1 0.3 0.7 0.1
implantations 14.2 14.3 14.3 15.1
# two females with implantation sites excluded due to sacrifice or death

Table 5 3 demonstrates that the mean numbers of corpora lutea, preimplantation losses, and implantation sites in the dose groups did not differ to a meaningful extent from the control group values indicating a homogeneous distribution regarding these parameters, whereas the fertility rate (percentage of inseminated females with implantations) was incidentally slightly lower at the 250 mg/kg level.

Gestation Rate
Table 5 4 and Section 8.7.1.6 in the Annex show the gestation rate (percentage of females with viable fetuses on day 21 p.c. of those with implantation sites).
Table 5 4: Gestation Rate
Dose Females with
viable fetuses on day 21 p.c. total resorption
mg/kg b.w./day n in % of females with implantations n
0 21 100.0 0
250 24 100.0 0
500 22 100.0 0
750 21 100.0 0

The gestation rate was unaffected by treatment at dose levels up to 750 mg/kg.

Mean Values of the Parameters of Intrauterine Development
Dose (mg/kg b.w./day) 0 250 500 750
number of females
with implantations (a) 21 24 22 21
with viable fetuses (b) 21 24 22 21
mean values per female
placental weight in g b 0.60 0.58 0.55 0.54
number of fetuses b 14.0 14.0 13.9 14.5
postimplantation loss a, b 0.2, 0.2 0.3, 0.3 0.5, 0.5 0.7, 0.7
males in % b 54.0 46.1 51.0 54.2
fetal weight in g b 4.87 4.63* 4.53** 4.19**
Statistically significant difference to control * = p < 0.05
Statistically significant difference to control ** = p < 0.01

Weight and Appearance of Placentas
Appearance and weights of placentas were unaffected by treatment at dose levels up to 750 mg/kg.
Postimplantation Loss, Number of Fetuses
Postimplantation loss and correspondingly the number of fetuses were unaffected by treatment at dose levels up to 750 mg/kg.
Sex of Fetuses
Fetal sex distribution was unaffected by treatment at dose levels up to 750 mg/kg.
Fetal Weight
The fetal weights were marginally to slightly decreased at dose levels of 250 mg/kg bw/day and above.

5.4.6 Fetal Malformations
Table 5 6 below gives an overview on the external, visceral, and skeletal malformations in the fetuses. A detailed description of the findings and the individual fetal data (malformations) are given in the Annex, Section 8.7.2.13, and an incidence table may be found in the Annex, Section 8.7.1.12. If fetuses revealed more than one malformation, the number of malformations is higher than the number of affected fetuses in these groups.
Table 5 6: Fetal Malformations
Malformation Dose (mg/kg b.w./day)
0 250 500 750
generalized edema 1 - - -
abdominal hernia with protruding organs 1 - - 1
lower jaw shortened 1 - - -
dilation of lateral, 3rd, 4th brain ventricles - - 1 -
1st sacral vertebral arch has the shape of a lumbar vertebral arch left (skeletal and cartilaginous parts), pelvis shift to caudal left with/without one lumbar vertebra missing - 1 1 -
scapula kinked, humerus bent, radius slightly bent bilateral (dysplastic)
- - - 1
additional cartilage at the 5th cervical vertebral arch left (has the shape of lamina ventralis), absence of lamina ventralis left (6th cervical vertebral arch), 6th cervical vertebral arch has the shape of a 7th cervical vertebral arch left, head of 1st ribs missing bilateral
- - - 1
rib(s) shortened 1 - - -
one supernumerary lumbar vertebra 1 1 - 1
cleft palate 1 - - -
number of fetuses per group 295 337 305 304
number of fetuses with malformations 4 2 2 4
malformed fetuses per group (%) 1.4 0.6 0.7 1.3
number of litters per group 21 24 22 21
number of litters with malformations 4 1 2 3
malformed litters per group (%) 19.0 4.2 9.1 14.3
() number of litters affected

The overall incidences of fetuses or litters with malformations lay within the range of historical control data, revealed no statistical significance, showed the highest values in the current control group and were thus unaffected by treatment at dose levels up to 750 mg/kg.
The types of malformations observed in this study are generally a representative sample of spontaneous malformations in the rat strain used (e.g. abdominal hernia with protruding organs, dilation of brain ventricle(s), malformation of the skeletal system) and comparable with spontaneous findings in the current control group and/or historical control groups.
At the 750 mg/kg level, one fetus revealed an abdominal hernia with protruding organs, for which a treatment related effect is not assumed due to its single occurrence, since this finding lay well within the range of historical control data of the rat strain used (see Annex, Section 8.8.10), and because this finding also occurred in the current control group. Furthermore, each one fetus of this dose level showed malformation of 5th-7th cervical vertebral arches with missing heads of 1st ribs bilateral, one supernumerary lumbar vertebra or dysplastic scapulae, humeri, and radii bilateral, for which a treatment related effect is not assumed due to their single occurrences, since these findings are known as spontaneous findings in the rat strain used (see Annex, Section 8.8.10), and because one supernumerary lumbar vertebra also occurred in the current control group. All remaining findings were also isolated findings, revealed no dose dependency and were thus also considered as incidental.
Thus, a treatment related effect on malformations (incidence or type) was not evident at dose levels up to 750 mg/kg.

Fetal External and Visceral Deviations
Table 5 7 below gives an overview on external and visceral deviations (findings other than malformations) in live fetuses. Individual data are listed in the Annex, and an incidence table may be found in the. The criteria for classifying the observed external or visceral findings as deviation or malformation are shown in the Annex, Section 8.6. If fetuses revealed more than one external or visceral deviation, the number of deviations is higher than the number of affected fetuses in these groups.
The overall incidences of fetuses or litters with external and visceral deviations were unaffected at dose levels up to 750 mg/kg and revealed the highest values in the 250 mg/kg group.
The external and visceral deviations observed in this study were of a common type and comparable with spontaneous findings in the current control group and/or historical control groups and represented the normal range of scattering in the rat strain used.


Table 5 7: Fetal External and Visceral Deviations
Deviation Dose (mg/kg b.w./day)
0 250 500 750
slight edema - 2 (1) 2 (2) -
retina slightly folded 1 1 - -
thyroid gland reduced in size - - 3 (2) -
thymus extended cranially 13 (10) 24 (13) 15 (11) 14 (9)
innominate artery missing 1 - - -
abdominal cavity filled with brown mass 1 1 6 (5) -
brown spot(s) in liver - 2 (2) 1 1
slight dilation of renal pelvis 23 (11) 19 (12) 12 (7) 11 (7)
dilation of renal pelvis 1 1 - -
slight dilation of ureter 16 (9) 16 (11) 11 (7) 9 (6)
dilation of ureter - 1 - -
testi(e)s lying slightly more cranially 8 (6) 9 (7) 9 (8) 4 (4)
testi(e)s lying more cranially - - - 1
testi(e)s reduced in size 1 - - -
right testis lying on the left side 1 - - 1
testi(e)s lying on bladder 4 (3) 10 (9) 2 (2) 9 (7)
brown mass in the area of salivary glands 1 1 - -
number of fetuses per group 295 337 305 304
number of fetuses with deviations 45 60 40 38
fetuses with deviat. per group (%) 15.3 17.8 13.1 12.5
number of litters per group 21 24 22 21
number of litters with deviations 17 21 19 18
litters with deviat. per group (%) 81.0 87.5 86.4 85.7
() number of litters affected

Thus, a treatment related effect on external and visceral deviations was not evident at dose levels up to 750 mg/kg.

5.4.8 Fetal Skeletal Deviations Including Cartilaginous Deviations
The individual fetal skeletal findings (including cartilaginous findings) are listed in the Annex, Section 8.7.2.12. An incidence table may be found in the Annex, Section 8.7.1.11. The criteria for classifying the observed skeletal findings as deviations (retardations, variations) or malformations are shown in the Annex, Section 8.6.
At the 750 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of several localizations (2nd distal phalanges of digits bilateral, 5th distal phalanges of digits right, 2nd-5th proximal phalanges of digits bilateral, 5th metacarpals bilateral, 1st and 5th distal phalanges of toes bilateral, 2nd-4th proximal phalanges of toes bilateral, 1st metatarsals bilateral, 5th sternebrae, 2nd-6th cervical vertebral bodies, 6th-8th caudal vertebral bodies), when calculation was done on a fetal basis and partially also on a litter basis, for which a treatment related effect is assumed, since fetal weights were also decreased at this dose level, and since nearly all values on a fetal basis lay outside the range of historical control data of the rat strain used. Furthermore an increased incidence of 14th ribs (punctiform left, comma shaped bilateral, sum) also occurred at this dose level, when calculation was done on a fetal basis, for which a treatment related effect cannot be excluded, because these values lay also outside the range of historical control data of the rat strain used.
At the 500 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of several localizations (5th sternebrae, 2nd-6th cervical vertebral bodies, calcaneus bilateral), when calculation was done on a fetal basis, for which a treatment related effect cannot totally be excluded, since fetal weights were also decreased at this dose level, and since nearly all values on a fetal basis lay outside the range of historical control data of the rat strain used (see Annex, Section 8.8.12). Statistically significantly progressed ossification of some localizations (5th distal phalanges of digits bilateral, 5th proximal phalanges of digits bilateral, parietal bones bilateral, interparietal bone) is considered to be incidental due to a lacking dose dependency, and since it is unlikely that retarded and progressed ossification occurs in parallel. Furthermore, an increased incidence of 14th ribs (punctiform left, sum) also occurred at this dose level, when calculation was done on a fetal basis, for which a treatment related effect cannot be excluded, because these values lay also outside the range of historical control data of the rat strain used..
At the 250 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of few localizations (2nd proximal phalanges of digits left, 2nd-4th proximal phalanges of toes bilateral, 1st metatarsals bilateral, 5th sternebrae, 3rd-6th cervical vertebral bodies, 8th caudal vertebral bodies), when calculation was done on a fetal basis, for which a treatment related effect cannot totally be excluded, since fetal weights were also decreased at this dose level, and since nearly all values on a fetal basis lay outside the range of historical control data of the rat strain used (see Annex, Section 8.8.12), although a dose dependency was lacking in some cases, and statistical significance was lacking in all but one case, when calculation was done on a litter basis. Furthermore, an increased incidence of flat 13th thoracic vertebral bodies occurred at this dose level, for which a treatment related effect is not assumed due to a lacking dose dependency.
Evaluation of fetal cartilaginous tissue revealed no treatment related findings at dose levels up to 750 mg/kg.
Thus, a treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) is assumed for retarded ossification of several localizations (digits, metacarpals, toes, metatarsals, sternebrae, cervical vertebral bodies, caudal vertebral bodies) at the 750 mg/kg level, and it cannot totally be excluded for retarded ossification of several localizations at the 500 mg/kg level and for borderline effects at the 250 mg/kg level in relation to dose-dependently though only marginally to slightly decreased fetal weights at all dose levels. Furthermore, a treatment related effect cannot be excluded for increased incidences of 14th ribs at the 750 mg/kg and 500 mg/kg levels.

Effect levels (fetuses)

Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Borderline effects at the 250 mg/kg on fetal body weight and degree of ossification.

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: Borderline effects at the 250 mg/kg on fetal body weight and degree of ossification.

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes

Any other information on results incl. tables

One female was sacrificed in moribund condition after it had shown signs of maternal toxicity. No further treatment related clinical findings occurred in the remaining females at dose levels up to 750 mg/kg bw/day.

Mean body weight loss occurred at the 750 mg/kg group between days 6-8 p.c., and absolute and corrected body weight gains were also decreased by treatment at dose levels of 500 mg/kg bw/day and above.

Food intake was decreased between days 6-12 p.c. at dose levels of 500 mg/kg and above, whereas water intake and fecal and urinary excretions were unaffected by treatment at dose levels up to 750 mg/kg bw/day.

One female of the 750 mg/kg group, which was sacrificed in moribund condition, revealed small and large intestines (including cecum) with gaseous contents at necropsy.

The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, and fetal sex distribution were unaffected by treatment at dose levels up to 750 mg/kg bw/day, whereas fetal weights were marginally to slightly decreased at dose levels of 250 mg/kg bw/day and above.

A treatment related effect on malformations and external and visceral deviations was not evident at dose levels up to 750 mg/kg.

A treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) is assumed for retarded ossification of many localizations (digits, metacarpals, toes, metatarsals, sternebrae, cervical vertebral bodies, caudal vertebral bodies) at the 750 mg/kg level, and it cannot totally be excluded for retarded ossification of several localizations at the 500 mg/kg and 250 mg/kg levels due to also decreased fetal weights at all dose levels. Furthermore, a treatment related effect cannot be excluded for increased incidences of 14th ribs at the 750 mg/kg and 500 mg/kg levels.

Applicant's summary and conclusion

Conclusions:
Twenty-five respectively thirty (250 mg/kg group, only) inseminated female Wistar rats each were treated daily orally by gavage with Dichlortoluol (purity 99.1 %) in polyethylene glycol 400 from day 6 to day 20 p.c. in the following doses: 0, 250, 500, and 750 mg/kg body weight (bw)/day, respectively. The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development.
Toxicity to the Females
One female of the 750 mg/kg group was sacrificed in moribund condition after it had shown signs of distinct maternal toxicity, and another female of this dose group was found dead most probably due to a misapplication. Except one further case of piloerection at this dose level, no further treatment related clinical findings occurred in the remaining females at dose levels up to 750 mg/kg bw/day.
Mean body weight loss occurred at the 750 mg/kg group between days 6-8 p.c., and absolute and corrected body weight gains were also decreased by treatment at dose levels of 500 mg/kg bw/day and above and resulted in statistically significantly reduced final body weights.
Food intake was decreased between days 6-12 p.c. at dose levels of 500 mg/kg and above, whereas water intake and fecal and urinary excretions were unaffected by treatment at dose levels up to 750 mg/kg bw/day.
One female of the 750 mg/kg group, which was sacrificed in moribund condition, revealed small and large intestines (including cecum) with gaseous contents at necropsy.
Toxicity to Intrauterine Development
The gestation rate was unaffected by treatment at dose levels up to 750 mg/kg.
Appearance and weights of placentas were unaffected by treatment at dose levels up to 750 mg/kg.
Postimplantation loss and correspondingly the number of fetuses were unaffected by treatment at dose levels up to 750 mg/kg.
Fetal sex distribution was unaffected by treatment at dose levels up to 750 mg/kg.
The fetal weights were marginally to slightly decreased at dose levels of 250 mg/kg bw/day and above.
A treatment related effect on malformations (incidence or type) was not evident at dose levels up to 750 mg/kg.
A treatment related effect on external and visceral deviations was not evident at dose levels up to 750 mg/kg.
A treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) is assumed for retarded ossification of several localizations (digits, metacarpals, toes, metatarsals, sternebrae, cervical vertebral bodies, caudal vertebral bodies) at the 750 mg/kg level, and it cannot totally be excluded for retarded ossification of several localizations at the 500 mg/kg level and for borderline effects at the 250 mg/kg level in relation to dose-dependently though only marginally to slightly decreased fetal weights at all dose levels. Furthermore, a treatment related effect cannot be excluded for increased incidences of 14th ribs at the 750 mg/kg and 500 mg/kg levels.
Summarizing and evaluating all data investigated the following no-observed-adverse-effect levels (NOAELs) were determined:
Maternal toxicity: 250 mg/kg bw/day
Developmental toxicity except fetuses: 750 mg/kg bw/day
Fetal weight and degree of ossification: < 250 mg/kg bw/day
Executive summary:

Twenty-five respectively thirty (250 mg/kg group, only) inseminated female Wistar rats each were treated daily orally by gavage with Dichlortoluol (purity 99.1 %) in polyethylene glycol 400 from day 6 to day 20 p.c. in the following doses: 0, 250, 500, and 750 mg/kg body weight (bw)/day, respectively. The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development.

Toxicity to the Females

One female of the 750 mg/kg group was sacrificed in moribund condition after it had shown signs of distinct maternal toxicity, and another female of this dose group was found dead most probably due to a misapplication. Except one further case of piloerection at this dose level, no further treatment related clinical findings occurred in the remaining females at dose levels up to 750 mg/kg bw/day.

Mean body weight loss occurred at the 750 mg/kg group between days 6-8 p.c., and absolute and corrected body weight gains were also decreased by treatment at dose levels of 500 mg/kg bw/day and above and resulted in statistically significantly reduced final body weights.

Food intake was decreased between days 6-12 p.c. at dose levels of 500 mg/kg and above, whereas water intake and fecal and urinary excretions were unaffected by treatment at dose levels up to 750 mg/kg bw/day.

One female of the 750 mg/kg group, which was sacrificed in moribund condition, revealed small and large intestines (including cecum) with gaseous contents at necropsy.

Toxicity to Intrauterine Development

The gestation rate was unaffected by treatment at dose levels up to 750 mg/kg.

Appearance and weights of placentas were unaffected by treatment at dose levels up to 750 mg/kg.

Postimplantation loss and correspondingly the number of fetuses were unaffected by treatment at dose levels up to 750 mg/kg.

Fetal sex distribution was unaffected by treatment at dose levels up to 750 mg/kg.

The fetal weights were marginally to slightly decreased at dose levels of 250 mg/kg bw/day and above.

A treatment related effect on malformations (incidence or type) was not evident at dose levels up to 750 mg/kg.

A treatment related effect on external and visceral deviations was not evident at dose levels up to 750 mg/kg.

A treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) is assumed for retarded ossification of several localizations (digits, metacarpals, toes, metatarsals, sternebrae, cervical vertebral bodies, caudal vertebral bodies) at the 750 mg/kg level, and it cannot totally be excluded for retarded ossification of several localizations at the 500 mg/kg level and for borderline effects at the 250 mg/kg level in relation to dose-dependently though only marginally to slightly decreased fetal weights at all dose levels. Furthermore, a treatment related effect cannot be excluded for increased incidences of 14th ribs at the 750 mg/kg and 500 mg/kg levels.

Summarizing and evaluating all data investigated the following no-observed-adverse-effect levels (NOAELs) were determined:

Maternal toxicity: 250 mg/kg bw/day

Developmental toxicity except fetuses: 750 mg/kg bw/day

Fetal weight and degree of ossification affected borderline: < 250 mg/kg bw/day