Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1985

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.3600 (Inhalation Developmental Toxicity Screen)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
; effects on preimplantation and late pregnancy were not examined (exposure on day 0-5 and day 16-20); weight of gravid uteri was not determined
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-dichlorobenzene
EC Number:
202-425-9
EC Name:
1,2-dichlorobenzene
Cas Number:
95-50-1
Molecular formula:
C6H4Cl2
IUPAC Name:
1,2-dichlorobenzene
Details on test material:
- Name of test material (as cited in study report): Orthodichlorobenzene
- Analytical purity: 98.81%
- Lot/batch No.: 01170.0
- Supplier: Standard Chlorine of Delaware, Inc.

Test animals

Species:
rat
Strain:
Fischer 344
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Mich.
- Weight at study initiation (at breeding): 175-220g
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: at least 2 weeks
- Animals were identified by unique ear tags

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 22°C
- Humidity (%): approximately 50%
- Photoperiod (hrs dark / hrs light): 12h light/12 h dark cycle

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: vaporised using pre-heated compressed air (120-135°C) and mixed and diluted in the chamber with incoming air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Rochester-type chamber (4.3m³) under dynamic airflow conditions
- Temperature, humidity, pressure in air chamber: temperature approximately 21°C; humidity approximately 50%
- Air flow rate: 800L air/min

- Samples taken from breathing zone: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
concentration in test chamber was verified at least every hour by IR-spectroscopy
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male to 1 female
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6 to 15 of gestation
Frequency of treatment:
6 h/d
Duration of test:
day 0 to 21 of gestation
No. of animals per sex per dose:
32 female (100 ppm)
30 female (200 ppm)
32 female (400 ppm)
Control animals:
other: the control group of rats was exposed to filtered room air for 6 h/d on days 6 through 15 of gestation
Details on study design:
At the end of the test the dams were sacrificed by carbon dioxid inhalation.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily; for indications of toxicity from the test material and adverse effects from exposure

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on day 6, 9, 12, 16 and 21 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- recorded at 3-day intervall starting on day 6

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: recorded at 3-day intervall starting on day 6

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 by carbon dioxide inhalation
- Organs examined: liver and kidney at time of cesarean section
Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number and position of resorption sites: Yes
- Number of live and dead fetuses
- Sex, body weight and crown-rump length of each fetus
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
body or organ weights
equality of variance: Bartlett's test (alpha=0.01)
analysis of variance: ANOVA (alpha=0.01)
if significant: Dunnett's test or Wilcoxon rank-sum test (with Bonferroni's correction)

frequency of fetal alterations, resorptions and population
basic unit of statistical analysis: litter
Wilcoxon rank-sum test (with Bonferroni's correction)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
all exposure levels: maternal toxicity, evident as significant decrease in mean body weight gains and slight decrease in food consumption;
exposure level 400 ppm: significant increase in both absolute and relative liver weight

2.4 mg/l group: significant increase in both absolute and relative (to body weight) liver weight

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEC
Effect level:
> 400 ppm
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
EMBRYO-/FETOTOXICITY:
no significant effects on litter size, resorption rate or fetal body weights; 

FETAL ALTERATIONS:
no significant increase in the incidence of major fetal malformations;
1.2mg/L: skeletal examination of rat fetuses: increase in the occurrence of spurs on the first lumbar vertebra and delayed ossification of sternebrae (effects not considered to be treatment related);
2.4mg/L:  significant increase in the occurrence of delayed ossification of cervical vertebral centra

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
> 400 ppm
Basis for effect level:
other: teratogenicity
Key result
Dose descriptor:
NOAEC
Effect level:
> 400 ppm
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Body weights and organ weights of pregnant rats exposed to 1,2-dichlorobenzene

 Dose  [ppm]  0  100  200  400
         
 No. of dams   27  27  28  28
 Maternal body weight on Gestation Day (mean ± SD) [g]         
 198 ± 8  202 ± 8  202 ± 8  203 ± 9
  9  202 ± 9  200 ± 9  198 ± 9  194 ± 7*
 12  212 ± 8  210 ± 8  208 ± 9  202 ± 8*
 16  229 ± 9  223 ± 9  220 ± 10* 214 ± 10* 
 21  268 ± 14  261 ± 16  263 ± 15  258 ± 19
         
 Maternal body weight gain on Gestation Days (mean ± SD) [g]         
 6-8  5 ± 5  -2 ± 6* -3 ± 4*  -8 ± 3*
 9-11   9 ± 4  10 ± 5  8 ± 3  8 ± 4
 12-15 17 ± 4  13 ± 4*  13 ± 7*  12 ± 5*
 16-20   39 ± 8  39 ± 10  42 ± 10  44 ± 12
 total 6-20  71 ± 13  59 ± 14*  60 ± 14*  56 ± 16*
         
 Maternal liver weight on Day 21         
 Absolute (mean ± SD) [g]   9.71 ± 0.61  9.90 ± 0.80  9.84 ± 0.76  10.25 ± 0.69*
 Relative (Organ weight [g]/ 100 g body wt (mean ± SD)  3.62 ± 0.19  3.79 ± 0.24* 3.74 ± 0.15   3.98 ± 0.23*
         
 Maternal kidney weight on Day 21        
 Absolute (mean ± SD) [g]   1.50 ± 0.11   1.51 ± 0.10   1.50 ± 0.12   1.47 ± 0.13
 Relative (Organ weight [g]/ 100 g body wt (mean ± SD)  0.56 ± 0.05   0.58 ± 0.06   0.57 ± 0.5   0.57 ± 0.08

* significantly different from the control value by Dunnett's test

Table 2: Observations made at the time of Cesarean section of bred rats exposed to 1,2-dichlorobenzene

Dose  [ppm]   0  100  200  400
         
 No. of bred females 32  32  30  32
 No. of maternal deaths  0  0  0  0
 % pregnant (visual or stained/ total bred)  84 (27/32)  87 (27/31) b  97 (29/30)  88 (28/32)
 No. of litters  27  27  28  28
 Corpora/lutea/dam c  11 ± 2  11 ± 2  11 ± 1  11 ± 2
Implantation sites/dam c  10 ± 2  9 ± 3  10 ± 2  10 ± 3
Fetuses/litter c  9 ± 2  9 ± 3 d  9 ± 2  9 ± 3 d
 Resorptions/litter c  0.46 ± 0.69  0.63 ± 0.93  0.54 ± 0.69  0.54 ± 0.64
 % implantations resorbed   5 (13/260)  7 (17/248)  6 (15/264)  6 (16/268)
 % litters with resorptions   37 (10/27)  44 (12/27)  46 (13/28)  50 (14/28)
 Litters totally resorbed   0 0
 Resorptions/litter with resorption   1.3 (13/10)  1.4 (17/12)  1.2 (15/13)  1.1 (16/14)
 Sex ratio, M:F  52:48  47:53  45:55 46:54 
 Fetal body weight [g] e  4.33 ± 0.42  4.42 ± 0.41  4.39 ± 0.15  4.47 ± 0.31
 Fetal crown-rump length [mm] e  42.90 ± 2.11 42.75 ± 1.94   42.90 ± 1.42  42.90 ± 1.64

No values were significantly different from the control value by a modified Wilcoxon test.

a : No. of females pregnant by visual inspection of the uterus or by sodium sulfide stain/ total bred.

b : Does not include one female which delivered a litter prior to cesarean section on Day 21 of gestation.

c : Mean ± SD.

d : Single fetuses in the 100 and 400 ppm groups were dead at the time of cesarean section.

e : Mean of litter means ± SD.

Table 3: Fetal alterations among litters of rats exposed to 1,2 -dichlorobenzene

Dose  [ppm]  0 100  200  400 
         
 No. of fetuses (litters) examined  247 (27)   231 (27)  249 (27)  252 (28)
 Visceral examination  132 (27)   126 (27)   132 (27) 137 (28) 
 Skeletal examination  247 (27)   231 (27)   249 (27)   252 (28)
 

 No. fetuses (litters) affected      

 
 Polydactyly  0  1 (1)
 Coarcted/ retroesophageal aortic arch 1 (1) 
Testicular agenesis, unilateral  0  1 (1)
 Microphtamia  0  1 (1)
 Vertebrae - delayed ossification a  60 (22)  60 (23)  54 (20)  79 (25)*
 Missing vertebrae and ribs  4 (1) b  0
 Cervical ribs  3 (2) b  3 (3)   1 (1)  1 (1)  
 Lumbar spurs a  1 (1)  1 (1)    6 (6) * 2 (2)  
 Sternebrae - delayed ossification a  66 (22)   83 (21) 84 (27)*  72 (23) 
 Major malformations (total) c  5 (2)  3 (3)    1 (1)   5 (3)
a : Not considered to be a major malformation. b : Two of the control fetuses had both missing vertebra (thoracic) and ribs and cervical ribs. c : Total number of fetuses (litters) which had one or more major malformations. * : Significantly different from control values by a modified Wilcoxon test.           

Applicant's summary and conclusion

Conclusions:
Inhalation of up to 400 ppm of 1,2-dichlorobenzene was not teratogenic in rats. The incidence of major malformations was not significantly increased in any of the exposed groups compared to control.
Executive summary:

Hayes WC et al., 1985

1,2-dichlorobenzene was tested for its ability to cause teratogenesis and fetotoxicity in pregnant female Fisher 344 rats similar to EU guideline B.31. This study was regarded as reliable with restrictions because of the following deviations: Effects on preimplantation and late pregnancy were not examined (exposure on day 0-5 and day 16-20); weight of gravid uteri was not determined. For this purpose, 30 to 32 bred female rats per group inhaled the vaporized test substance at dose levels of 100, 200 and 400 ppm in Rochester-type chambers for 6 h/d on day 6 to 15 of gestation.

As an negative control 32 bred females inhaled merely air following the above mentioned procedure.

The concentration of the test substance in the chamber was checked by IR-spectrometry. The maternal animals were observed daily for toxicity and adverse effects throughout the test period. Body weights were recorded on day 6, 9, 12, 16 and 21 of gestation.

Food and water consumption was recorded at 3-day intervals starting on day 6.

Test animals were sacrificed on day 21 and the uterine horns were exteriorized and the maternal livers and kidneys weighed. Following data were recorded: number of fetuses (dead and alive), number of resorption sites, number of corpora leutea, sex, body weight and crown-rump length of each fetus, any gross external alteration. Uteri of non-pregnant animals were stained and implantation sites examined. One half of each litter (randomized) was examined for soft tissue alterations and all heads were examined for skeletal alterations.

According to the results of the present study the inhalation of 1,2-dichlorobenzene in a dose of up to 400 ppm for 6 h/d during day 6 to 15 of gestation did not cause significant teratogenicity or fetotoxicity in female Fisher 344 rats. The incidence of malformation was not significantly increased

Thus, under the conditions tested here, the test substance 1,2-dichlorobenzene does not have any significant teratogenic or fetotoxic effect on organogenesis in F344 rats.

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