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REACH

Dichloromethylbenzene

EC number: 249-854-8 | CAS number: 29797-40-8

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is no Reproduction/ Developmental Screening Test available for Dichlorotoluene Mixture. According to REACH Commission Regulation (EU) 2015/283 the Regulation (EC) No 1907/2006 is amended as follows: This study does not need to be conducted if a pre-natal developmental toxicity study (Annex IX, 8.7.2) or, either an Extended One-Generation Reproductive Toxicity Study (B.56, OECD TG 443) (Annex IX, section 8.7.3) or a two-generation study (B.35, OECD TG 416), is available. These requirements are fulfilled.

An Extended One-Generation Reproductive Toxicity Study with Dichlorotoluene Mixture by daily gavage in the rat according to OECD TG 443 was conducted following an ECHA decision on a compliance check. The study was requested by the ECHA Decision number TPE D 2114394001-60-01/F.

The objective of this study was to evaluate the pre- and post-natal effects of Dichlorotoluene Mixture, an industrial chemical, when administered orally, by gavage, to Han Wistar rats. The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring. The study was requested by the ECHA Decision number TPE D 2114394001-60-01/F.

Based on the results of this study a No Observed Adverse Effect Level (NOAEL) for systemic toxicity in F0 and F1 male rats, when treated with Dichlorotoluene Mixture was 50 mg/kg/day, due to hyaline droplets with associated degenerative changes (basophilic tubules and granular casts). This alpha 2u globulin associated nephropathy was considered adverse at 150 or 300 mg/kg/day. However, it is acknowledged that this finding is specific to the male rat and is generally considered to be of no relevance to man. Excluding this finding the NOAEL for male rats is 300 mg/kg/day.

The NOAEL for systemic and reproductive toxicology in adult female rats was concluded to be 300 mg/kg/day. The dose of 300 mg/kg/day is also concluded to be the NOAEL for the offspring based on the low observed body weight gain following weaning.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 300 mg/kg bw/day
Study duration:: subchronic
Species:: rat
Quality of whole database:: guiedline study according OECD TG 443.

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

In an Extended One-Generation Reproductive Toxicity Study according to OECD TG 443, in the F0 generation, three groups of 25 male and 25 female Han Wistar rats received Dichlorotoluene Mixture at doses of 50, 150 or 500/300 mg/kg bw/day (treatment to Group 4 (500 mg/kg bw/day) was lowered to 300 mg/kg bw/day from Week 11 (commencement of pairing)) at a volume dose of 5 mL/kg/day. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. In the F1 generation, 20 males and 20 females were treated at 50, 150 or 300 mg/kg bw/day from weaning to their scheduled termination, at the same volume-dose as the F0 generation. A similarly constituted Control group received the vehicle, polyethylene glycol 400 (specific gravity 1.125), at the same volume dose throughout the same period.

The study was requested by the ECHA Decision number TPE‑D‑2114394001-60-01/F.

F0: Data were recorded on clinical condition, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance. Clinical pathology (hematology, blood chemistry and thyroid‑related hormones), sperm assessment, organ weight, macroscopic pathology and microscopic pathology investigations were performed.

The F1 generation comprised of two cohorts. Each cohort comprised of 20 male and 20 female progeny from each dose group and were treated with Dichlorotoluene Mixture, by oral gavage administration, at doses of 0 (Control), 50, 150 or 300 mg/kg bw/day from weaning (Day 21 of age) until scheduled sacrifice. Cohort A was terminated at approximately Week 13 of age and Cohort B was terminated at approximately Week 14 of age.

F1: Offspring, data were recorded on clinical condition, litter size and survival, sex ratio, body weight, anogenital distance, nipple count (male offspring on Day 13 of age), organ weights and macropathology (on Day 4 or 22 of age), were assessed. Blood samples were also collected from selected offspring on Day 4 and 22 of age for investigation of thyroid-related hormones.

F1: Cohort A, data were recorded on clinical condition, body weight, food consumption, sexual maturation, vaginal opening and estrous cycles. Clinical pathology (hematology, blood chemistry, urinalysis and thyroid-related hormones), sperm assessment, ovarian follicle counts, organ weight, macroscopic pathology, microscopic pathology and immunophenotyping investigations were performed.

F1: Cohort B, data was recorded on clinical condition, body weight, food consumption, sexual maturation, selected organ weights and a targeted set of macroscopic pathology investigations were performed.

Results

F0 adults and F1 offspring up to day 25 of age

Treatment at 500 mg/kg/day was not tolerated by a small number of animals. Four animals died or were killed for welfare reasons within the ten week pre‑pairing treatment period following brief periods of respiratory distress and two had blocked nasal turbinates. Treatment at 500 mg/kg/day was reduced to 300 mg/kg/day from Week 11; Day 1 (day of pairing). One female receiving 150 mg/kg/day was killed for welfare reasons following signs of respiratory distress.

There were no systemic clinical signs considered to be related to treatment and no effect on body weight gain during the 18 week treatment period in males and 17 week treatment period in females, including gestation and lactation to Day 21 post-partum. Food consumption was unaffected by treatment.

Estrous cycles, pre‑coital interval, mating performance, fertility, or gestation length and gestation index; the mean number of implantations and litter size, the ratio of males to females or survival indices were unaffected by treatment.

Minor findings in the hematology or blood plasma parameters had no microscopic correlate.

Following parental treatment at 50, 150 or 500/300 mg/kg/day, the clinical condition of the F1 pups remained good. Mean body weights of treated male and female offspring on Day 1 of age were marginally low, without relationship to dose. Subsequent body weight gain was marginally lower than Control and, at formal commencement of the F1 generation, mean body weights were low at 50, 150 or 300 mg/kg/day. Body weight gain improved in males and females at 50 or 150 mg/kg/day and females at 300 mg/kg/day; however, body weight gain in males at 300 mg/kg/day remained marginally, low and was considered adverse.

In the F1, anogenital distance was unaffected by parental treatment at 50, 150 or 500/300 mg/kg/day and males did not develop nipples. Serum T4 and TSH concentrations in the adult and offspring on Days 4 and 22 of age were unaffected by treatment.

There was no effect of treatment on the number or motility or morphology of the sperm.

There were no macroscopic findings attributed to treatment.

Selected F1 offspring - Cohorts 1A and 1B

The clinical condition of the F1 animals remained generally good.

Six animals receiving 300 mg/kg/day and two receiving 150 mg/kg/day died, or were killed for welfare reasons, after showing signs of respiratory distress and one animal receiving 150 mg/kg/day was mis-dosed.

Sexual maturation of the F1 and the period between vaginal opening and first estrus, and estrous cycles at Week 11 of age (before termination) were unaffected by treatment at 50, 150 or 300 mg/kg/day.

Serum T4 and TSH concentrations in the adult and Immunophenotyping parameters, measured in spleen leukocytes (both Cohort 1A), were unaffected by treatment at 50, 150 or 300 mg/kg/day.

Pathology of the F0 and F1 in males only

Microscopic examination revealed that oral gavage administration of Dichlorotoluene Mixture resulted in test item-related changes in the liver and kidneys ofF0 and F1 males. F0 and F1 females were unaffected. In the liver, incidences of centrilobular hypertrophy were dose-related in males treated at 150 or 500/300 or 300 mg/kg/day and correlated with increased liver weights in F0 males. This possibly resulted from disturbance of hepatic metabolic activity and was therefore considered an adaptive response. In the kidney, there was an accumulation of hyaline droplets in the cytoplasm of the cortical tubular epithelium, accompanied by basophilic tubules, and/or granular casts at 150 or 500/300 mg/kg/day. This showed dose-relationship and were present in the kidneys of F0 and F1 (Cohort A and B) males given 150 or 500/300 or 300 mg/kg/day, and F0 and F1 (Cohort A) males given 50 mg/kg/day. These changes correlated with increased kidney weights seen in F0 males and males given 300 mg/kg/day of the F1 (Cohort A). Hyaline droplets are a common finding in male kidneys, composed of alpha-2u-globulin and in more severe cases, can be associated with degenerative changes (basophilic tubules and granular casts), as seen in this study. Hyaline droplet-mediated nephropathy is considered rat specific and therefore not relevant to humans as little or no alpha-2u-globulin is present in humans.

Conclusion

Based on the results of this study a No Observed Adverse Effect Level (NOAEL) for systemic toxicity in F0 and F1 male rats, when treated with Dichlorotoluene Mixture was 50 mg/kg/day, due to hyaline droplets with associated degenerative changes (basophilic tubules and granular casts). This alpha‑2u‑globulin associated nephropathy was considered adverse at 150 or 300 mg/kg/day. However, it is acknowledged that this finding is specific to the male rat and is generally considered to be of no relevance to man. Excluding this finding the NOAEL for male rats is 300 mg/kg/day.

Special consideration should be made when administering Dichlorotoluene Mixture (classified for skin irritancy) by the oral gavage route, due to the low incidence of respiratory distress and death observed at 150 or 300 mg/kg/day.

Effects on developmental toxicity

Description of key information

Two valid developmental toxicity studies according to OECD TG 414 are available in rats and rabbits for Dichlorotoluene Mixture.

In these studies in rats and rabbits no treatment related effect on malformations (incidence or type) and external and visceral deviations was evident at any dose tested. In the rat study fetal toxicity (weight and degree of ossification) was seen with borderline to slight effects starting at the low dose, 250 mg/kg/day. No fetal toxicity was observed in the rabbit study up to the highest dose tested (250 mg/kg/day).

Link to relevant study records

Referenceopen all close all

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

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.

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.

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

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):

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):

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

Number of abortions:

no effects observed

Description (incidence and severity):

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Early or late resorptions:

no effects observed

Description (incidence and severity):

Dead fetuses:

no effects observed

Description (incidence and severity):

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

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.

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

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.

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.

Abnormalities:

effects observed, treatment-related

Localisation:

other: Borderline effects at the 250 mg/kg on fetal body weight and degree of ossification.

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

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.

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.

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:

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.

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

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Rabbit: female albino rabbits, New Zealand White (NZW) strain (SPF-Quality), from a non-inbred laboratory colony. Nulliparous, non-pregnant and untreated females

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% Aqueous carboxymethyl cellulose (carboxymethyl cellulose) with 0.1% Tween-80.

Details on exposure:

Method: Oral gavage, using a plastic catheter attached to a plastic disposable syringe. Formulations were placed on a magnetic stirrer during dosing.

Frequency: Once daily for 7 days per week, approximately the same time each day. Animals were dosed daily within 3 hours of formulation and with a
maximum of 6 hours difference between the earliest and latest dose over the entire dosing period.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Details on mating procedure:

One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.

Duration of treatment / exposure:

From days 6 to 28 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day. Animals were dosed daily within 3 hours of formulation and with a maximum of 6 hours difference between the earliest and latest dose over the entire dosing period.

Duration of test:

Day of necropsy: Day 29 post-coitum.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

40 mg/kg bw/day

Remarks:

Dichloromethylbenzene

Dose / conc.:

100 mg/kg bw/day

Remarks:

Dichloromethylbenzene

Dose / conc.:

250 mg/kg bw/day

Remarks:

Dichloromethylbenzene

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Maternal examinations:

Mortality / Viability: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ (2000)7). The circumstance of any death was recorded in detail.

Clinical signs: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. Cage debris was examined to detect abortion or premature birth, if applicable.

Body weights: Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum.

Food consumption: Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths (early and late resorptions).
- The weight of each fetus.
- The sex of each fetus (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.

Animals sacrificed before planned necropsy, were subjected to relevant examinations of the ovaries and uterine horns. Findings were given in the individual data, but not in the summary tables.

Fetal examinations:

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that were considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized by administration of approximately 0.3 mL (=60mg) of sodium pentobarbital into the oral cavity using a small flexible plastic or metal feeding tube. Nonviable fetuses (the degree of autolysis is minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed (if possible).

Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe 1984. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar 1972. The sex of all fetuses was determined by internal examination.

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution. Tissues were then transferred to a 70% aqueous ethanol for subsequent processing and soft-tissue examination using the Wilson 1965 sectioning technique. After examination, the tissues were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of (all groups) were examined by a mid-coronal slice.

All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol for subsequent staining of the skeletons.

Skeletal:
The fetuses from all groups were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson 1926. Subsequently, the skeletal examination was done first on all fetuses from Groups 1 and 4. Due to the relative low number of litters (n=15) available in Group 4, it was decided in consultation with the Sponsor to extent skeletal examination to all fetuses from Group 3.

The specimens are archived in glycerin with bronopol as preservative.

A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

The following definitions were applicable for implantation data:
- Fetal (late) resorptions were defined as a dead fetus with external degenerative changes and presence of distinguishable features such as head or limbs.
- Embryonic (early) resorptions were defined as evidence of implantation without presence of distinguishable features such as head or limbs.
- Dead fetus was defined as a non-viable fetus without external degenerative changes and presence of distinguishable features such as head or limbs.
- Post-implantation loss included embryonic (early) resorptions, fetal (late) resorptions and dead fetuses.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 5) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (Ref. 6 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 7) was applied to frequency data.
- The Mann Whitney test (Ref. 8) was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 9) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Ref. 10) was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A total of six females at 250 mg/kg/day did not survive until scheduled necropsy (Day 29 post-coitum). Five females were killed in extremis between Days 22-26 post-coitum. Another female had an abortion and was euthanized on Day 28 post-coitum. All six females were noted with lethargy (slight to moderate degree), abnormal licking, hunched posture, pale skin (slight degree), piloerection, reduced production of (pale) feces (slight to severe degree) and/or a lean appearance. In addition, red fluid was found on the manure tray of the cage of one female on Days 25 and 26 post-coitum. All these females had severely reduced to no food intake from Days 13-16 post-coitum onwards and significantly body weight loss (10-16%). In addition there was a higher incidence and severity of reduced feces production in the 250 mg/kg/day group compared to the control group and other treated groups. Females in this dose
group with highest toxicity had pale feces as well.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food efficiency:

not specified

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

not specified

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:

no effects observed

Description (incidence and severity):

There were no gross findings at necropsy that were considered to be toxicologically relevant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

There were no maternal findings at 40 and 100 mg/kg/day that were considered to be toxicologically relevant.

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One high dose female (no. 73) had an abortion on Day 28 post-coitum and was euthanized. She delivered 12 dead fetuses. There were 3, 3, 3 and 1 non-pregnant females in the control, 40, 100 and 250 mg/kg/day groups. All remaining females were pregnant. As in the high dose group also one female (no. 73) had an abortion and 5 other females (71, 72, 77, 80, 85) were euthanized preterm, the number of litters available for fetal evaluation was 19, 19, 19 and 15, respectively.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no effects on the number of pregnant females, number of corpora lutea, implantation sites, or pre- or post-implantation loss noted with treatment up to 250 mg/kg.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no effects on the number of pregnant females, number of corpora lutea, implantation sites, or pre- or post-implantation loss noted with treatment up to 250 mg/kg.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no effects on the number of pregnant females, number of corpora lutea, implantation sites, or pre- or post-implantation loss noted with treatment up to 250 mg/kg.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

One high dose female (no. 73) had an abortion on Day 28 post-coitum and was euthanized. She delivered 12 dead fetuses.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no effects on the number of pregnant females, number of corpora lutea, implantation sites, or pre- or post-implantation loss noted with treatment up to 250 mg/kg.

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Rational for dose selection and maternal toxicity in pilot study:
In order to set the dose levels for the main teratology study, a dose range finding study was performed. Four groups of 6 females were exposed to 100, 250 and 500 mg/kg for Days 6 to 28 post-coitum inclusive by oral gavage.

Maternal findings
At 500 mg/kg, no animals survived until Day 29 post-coitum.
Three females were killed in extremis due to their poor condition. Female no. 19 was euthanized on Day 14 post-coitum (last dosing the day before), and female nos. 21 and 22 were euthanized on Day 21 post-coitum. These animals had lost 12-18% of their weight since start of treatment on Day 6 post-coitum, and had reduced consumption of water or food (up to severe). Clinical signs consisted of lethargy (slight to moderate), slow breathing, a pale and/or lean appearance, hunched posture, piloerection, and/or reduced production of (pale) feces (up to severe). In addition, abnormal licking of the lips was seen immediately after dosing. Gross findings at necropsy included emaciation (no. 19), and reduced thickness and/or gelatinous appearance of the wall between cardia and pyleryus of the stomach (nos. 21 and 22). Female no. 19 had 8 normal implantations in development, female nos. 21 and 22 had respectively 8 and 12 dead fetuses in their uterus.
As from these data it was obvious that dichloromethylbenzene at a dose of 500 mg/kg was too toxic to be tested in the subsequent main teratology study, treatment of the remaining females (nos. 20, 23 and 24) was stopped after dosing on Day 21 post-coitum and animals were euthanized the day thereafter. Clinical signs consisted of abnormal licking of the lips seen immediately after dosing, reduced production of (enlarged) feces (up to severe). These females had reduced food consumption and up to 5% body weight loss from Days 6-9 post-coitum, followed by (partial) recovery to values considered to be in the normal range. At necropsy, the colon of one female (no. 24) was noted to be distended with gas. Another female (no. 23) had a watery-clear cyst on her left oviduct, which was considered as a background finding. All three females were pregnant with respectively 7, 7 and 11 live fetuses.

There were no unscheduled deaths in the control, 100 and 250 mg/kg group, respectively.
Like in the high dose group, animals at 100 and 250 mg/kg showed abnormal licking of the lips after dosing, though incidence and onset of this sign increased slightly with increasing doses.
There were no other clinical signs noted at 100 and 250 mg/kg that were considered to be treatment related.

Slightly reduced food intake (absolute and relative to body weight) was noted at 250 mg/kg as compared to controls from Days 6-20 post-coitum. Changes reached statistical significance from Days 6-9 and 13-16 post-coitum.

At 100 and 250 mg/kg, body weight and body weight gain were within the normal range of biological variation, except for one female (no. 13) at 250 mg/kg which lost 9% of her weight from Days 26-29 post-coitum. She was noted emaciated at necropsy.

One female of control group was non-pregnant. All other females of the control, 100 and 250 mg/kg group were pregnant with viable fetuses.

There were no effects on the number of pregnant females, number of corpora lutea, implantation sites, or pre- or post-implantation loss noted with treatment up to 250 mg/kg. All values remained within the normal range of biological variation. Females at 500 mg/kg (all pregnant) were not available for evaluation as they were euthanized preterm. For further details, see above.

Fetal findings
Mean fetal body weights, mean litter size, number of viable fetuses (% per litter), and early and late resorptions (% per litter) were considered unaffected by treatment up to 250 mg/kg. No external malformations were noted up to 250 mg/kg. No litters were available at 500 mg/kg. All dams were euthanized preterm.

Based on the results of this dose range finding study, selected dose levels for the main study (Project 508049) were 40, 100 and 250 mg/kg.
------------------------------------
Observations in the main study:
A total of six females at 250 mg/kg did not survive until scheduled necropsy (Day 29 post-coitum). Five females were killed in extremis between Days 22-26 post-coitum. Another female had an abortion and was euthanized on Day 28 post-coitum. All six females were noted with lethargy (slight to moderate degree), abnormal licking, hunched posture, pale skin (slight degree), piloerection, reduced production of (pale) feces (slight to severe degree) and/or a lean appearance. In addition, red fluid was found on the manure tray of the cage of one female on Days 25 and 26 post-coitum. All these females had severely reduced to no food intake from Days 13-16 post-coitum onwards and significantly body weight loss (10-16%).

In addition there was a higher incidence and severity of reduced feces production in the 250 mg/kg group compared to the control group and other treated groups. Females in this dose group with highest toxicity had pale feces as well.

Animals at 250 mg/kg showed, on average, slight body weight loss (up to 3%) until Day 23 post-coitum, followed by partial recovery thereafter. However, at the individual level differences were relatively high with the largest increase in body weight reaching 12% and largest body weight loss of 16%. Corrected body weight gain (for uterus) was unaffected by treatment up to 250 mg/kg.

Animals at 250 mg/kg had a reduced mean food consumption (before or after correction for body weight) between Days 6-20 post-coitum.

Overall, the MTD is clearly reached/exceeded at the high dose.

There were no maternal findings at 40 and 100 mg/kg that were considered to be toxicologically relevant.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

effects observed, treatment-related

Description (incidence and severity):

A total of six females at 250 mg/kg did not survive until scheduled necropsy (Day 29 post-coitum).

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no toxicologically relevant changes in fetal body weights up to 100 mg/kg/day.
At 250 mg/kg/day, mean male and female fetal body weights (and of both sexes combined) appeared lower than control levels (both sexes combined: 37.6 grams at 250 mg/kg/day vs. 39.8 grams in the control group), without achieving a level of statistical significance. All values remained within the normal range of biological variation.
At 40 and 100 mg/kg/day, mean fetal body weights (males and females separately and both sexes combined) were considered unaffected by treatment (both sexes combined: 41.8 and 39.5 grams, respectively).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Litter sizes were not affected by treatment up to 250 mg/kg/day.
Mean litter sizes (viable fetuses) were 8.1, 7.9, 8.7 and 8.7 for the control, 40, 100 and 250 mg/kg/day group, respectively. These numbers remained within the normal range of biological variation.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no treatment related effects on the sex ratio between control and treated animals.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to 250 mg/kg/day.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment up to 250 mg/kg/day.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 250 mg/kg/day.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
Litter size
Litter sizes were not affected by treatment up to 250 mg/kg.
Mean litter sizes (viable fetuses) were 8.1, 7.9, 8.7 and 8.7 for the control, 40, 100 and 250 mg/kg group, respectively. These numbers remained within the normal range of biological variation. The number of dead fetuses was 2, 1, 5 and 0 in the control, 40, 100 and 250 mg/kg group, respectively. All 5 fetuses in the mid dose group were from the same litter. They were next to 5 live fetuses. As their dam was noted with severe findings during in-life, the death of these fetuses was considered to be secondary to maternal toxicity. Of note is that at scheduled necropsy on Day 29 post-coitum one placenta was found to be larger than normal and discolored dark-red. This may be indicative for an impaired placental function.

Sex ratio:
There were no treatment related effects on the sex ratio between control and treated animals.

Fetal body weight:
There were no toxicologically relevant changes in fetal body weights up to 100 mg/kg/day.

At 250 mg/kg, mean male and female fetal body weights (and of both sexes combined) appeared lower than control levels (both sexes combined: 37.6 grams at 250 mg/kg vs. 39.8 grams in the control group), without achieving a level of statistical significance. All values remained within the normal range of biological variation. At 40 and 100 mg/kg, mean fetal body weights (males and females separately and both sexes combined) were considered unaffected by treatment (both sexes combined: 41.8 and 39.5 grams, respectively).

Fetal morphological examinations:
External malformations and variations
There were no treatment related effects on external morphology following treatment up to 250 mg/kg.

The only external malformation in a treated group occurred at 250 mg/kg in fetus A086-08. This fetus had an omphalocele, that was also observed in control fetus A014-08 and thus was not considered to be treatment related. Another control fetus (A003-09) was observed with carpal flexure (without any apparent skeletal origin). There were no other external abnormalities in this study.

There were no external variations seen for any fetus in any group.

Visceral malformations and variations
There were no treatment related effects on visceral morphology following treatment up to 250 mg/kg.

Visceral malformations were noted in 3(3), 2(2), 2(2) and 2(2) fetuses (litters) in the control, 40, 100 and 250 mg/kg groups, respectively. At 250 mg/kg, these were tetralogy of Fallot (A075-11) and malpositioned kidney (A079-03). At 100 mg/kg, these were absent (accessory) lung lobe (A057-07) and abnormal consistency of gallbladder content (gallstone; A050-06). At 40 mg/kg, these were combined cases of atrial septum defect, dilated aortic arch and atretic pulmonary trunk (A035-07, A038-11), and in the control group, two cases of absent (accessory) lung lobe (A003-09, A014-07) and one with folded retina (A010-03) were observed. Besides, two dead fetuses in one control litter (A010-02 and A010-04) had multiple cardiovascular abnormalities. The visceral malformations noted were not considered to be treatment-related as they occurred singly or at a very low incidence, were distributed randomly across treated groups, occurred at frequencies that were within the normal range of biological variation or were seen for a control fetus only.
Visceral variations were noted for 7.7%, 9.5%, 7.7% and 15.9% of the fetuses per litter in the control, 40, 100 and 250 mg/kg groups, respectively. The total percentage per litter in Group 4 is increased compared with the control value (not statistically significantly). This is not considered to be caused by treatment, but to be a consequence of the low number of litters available in Group 4 in combination with the presence of litter A087 in that group. This litter had only one fetus which was observed with a partially undescended thymus horn, causing an increased group mean litter proportion of that finding (8.5% versus control mean value 0.0% per litter) and hence a relatively high total litter percentage of visceral variations at 250 mg/kg.
Remaining visceral variations noted were not considered to be treatment related as they occurred across all groups, at a very low incidence and/or in the absence of a dose-dependent distribution.

Skeletal malformations and variations
There were no treatment related effects on skeletal morphology following treatment up to 250 mg/kg.

Three fetuses out of two litters in the high dose group (250 mg/kg) had a skeletal malformation, namely fusion of sternebrae. These were fetuses A068-07, -09 and A069-02. Because of this low incidence and because it was also noted in a control litter in two fetuses (A010-03 and dead fetus A010-04) and in a single Group 3 fetus (A047-07), this malformation was not considered to be treatment related.

The only other malformation in this study, a vertebral anomaly with associated rib anomaly, was observed in two Group 3 fetuses (A057-07 and A064-11) at an incidence below the upper historical control value (1.1% versus 2.8%) and thus was not considered to be related to treatment.

Of the skeletal variations, the only noteworthy finding was the occurrence of 13th full ribs at 250 mg/kg. This finding was noted at a statistically significantly increased incidence of 73.8% per litter against concurrent control value of 47.0 % per litter. The value at 100 mg/kg was 42.8% per litter. However, because the value at 250 mg/kg was only slightly above the upper historical control limit (71.4% per litter) and 13th full ribs are the most common fetal skeletal variation amongst historical controls, this finding was not considered to be treatment related.

Remaining skeletal variations were not considered treatment related as they occurred infrequently, occurred at frequencies that were within the range of available historical control data or were observed in control fetuses only.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

Conclusions:

Maternal findings:
Maternal toxicity was noted at 250 mg/kg/day.
Developmental findings:
There were no toxicologically relevant effects on the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal developmental malformations or variations) with treatment up to 250 mg/kg/day.

Executive summary:

In conclusion, based on the results of this prenatal developmental toxicity study, the following No Observed Adverse Effect Levels (NOAELs) for dichloromethylbenzene were established:

Maternal NOAEL: 100 mg/kg (with severe toxicity including mortility at 250 mg/kg/day).

Developmental NOAEL: 250 mg/kg; the highest dose tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: LOAEL
: 250 mg/kg bw/day
Species:: rat
Quality of whole database:: Developmental toxicity study according to OECD TG 414

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

Developmental toxicity studies according to OECD TG 414 are available in rats and rabbits for Dichlorotoluene Mixture.

Rats (Langewische 2013):

Female Wistar rats were treated daily by gavage with test compound in polyethylene glycol 400 from day 6 to day 20 p.c. with the following doses: 0, 250, 500, and 750 mg/kg/day. 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.

One female of the 750 mg/kg/day group was sacrificed in moribund condition after it had shown signs of distinct maternal toxicity and revealed small and large intestines (including cecum) with gaseous contents at necropsy. 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/day. Mean body weight loss occurred at the 750 mg/kg/day 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/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/day and above, whereas water intake and fecal and urinary excretions were unaffected by treatment at dose levels up to 750 mg/kg/day. Overall, the NOAEL for maternal toxicity was 250 mg/kg/day.

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/day, whereas fetal weights were marginally to slightly decreased at all dose levels; 250 mg/kg/day and above.

A treatment related effect on malformations (incidence or type) and external and visceral deviations was not evident at dose levels up to the highest dose tested: 750 mg/kg/day.

Rabbits (Peter 2015):

New Zealand White rabbits were dosed with the test item in 1% carboxymethyl cellulose + 0.1% Tween-80 by daily oral gavage from days 6 to 28 post-coitum at doses of 40, 100 and 250 mg/kg/day. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. Investigation was performed on general tolerance of the test substance by the females. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.

A total of six females in the high dose group, 250 mg/kg/day, did not survive until scheduled necropsy. Five females were killed in extremis between Days 22-26 post-coitum. Another female had an abortion and was euthanized on Day 28 post-coitum. Overall, the MTP is clearly exceeded in the high dose group based on the high mortality 6/21 (28.6% mortality).

There were no toxicologically relevant effects on the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal developmental malformations or variations) with treatment up to 250 mg/kg.

Of the skeletal variations, the only noteworthy finding was the occurrence of 13th full ribs at 250 mg/kg. This finding was noted at a statistically significantly increased incidence of 73.8% per litter against concurrent control value of 47.0 % per litter. The value at 100 mg/kg was 42.8% per litter. However, because the value at 250 mg/kg was only slightly above the upper historical control limit (71.4% per litter) and 13th full ribs are the most common fetal skeletal variation amongst historical controls and taken together with high maternal mortality in this high dose group (6/21) this finding was considered to be not treatment related and/or not toxicologically relevant.

Justification for classification or non-classification

Based on the results of the Extended One-Generation Reproductive Toxicity Study with Dichlorotoluene Mixture by daily gavage in the rat according to OECD TG 443, a No observed Adverse Effect Level (NOAEL) for systemic toxicity in F0 and F1 male rats was 50 mg/kg/day, due to hyaline droplets with associated degenerative changes (basophilic tubules and granular casts). This alpha 2u globulin associated nephropathy was considered adverse at 150 or 300 mg/kg/day. However, it is acknowledged that this finding is specific to the male rat and is generally considered to be of no relevance to man. Excluding this finding the NOAEL for male rats is 300 mg/kg/day (highest applied dose).

The NOAEL for systemic and reproductive toxicology in adult female rats was concluded to be 300 mg/kg/day. The dose of 300 mg/kg/day (highest applied dose) is also concluded to be the NOAEL for the offspring based on the low observed body weight gain following weaning.

In developmental toxicity studies in rats and rabbits no treatment related effect on malformations (incidence or type) and external and visceral deviations was evident at any dose tested. In the rat study fetal toxicity (weight and degree of ossification) was seen with borderline to slight effects starting at the low dose, 250 mg/kg/day. No fetal toxicity was observed in the rabbit study up to the highest dose tested (250 mg/kg/day). Dichlorotoluene Mixture is considered to be a not developmental toxicant.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification for toxicity to reproduction is not justified.

Additional information

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Registration Dossier

Registration Dossier

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Diss Factsheets

Dichloromethylbenzene

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

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Reference 1

Reference 2

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Justification for classification or non-classification

Additional information

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