2-chlorotoluene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well documented and scientifically acceptable - comparable to guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Materials and methods

Principles of method if other than guideline:

From gestation days (GD) 6 to 19, 25 animals/dose group were transferred daily to exposure chambers and exposed to a an atmosphere of the test material at concentrations of 0, 1, 3, or 9 mg/litre air for 6 hours. All animals were killed on day 20 of pregnancy, dissected and examined for abnormalities and changes in maternal organs. The ovaries and uteri were examined to determine number of corpora lutea, number of distribution of live young, number and distribution of embryonic/foetal deaths, individual foetal weight, gross foetal abnormalities. half of the pups in each litter were preserved for subsequent sectioning to discover visceral abnormalities; the remainder were fixed for skeletal examination.

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

2-chlorotoluene

EC Number:

202-424-3

EC Name:

2-chlorotoluene

Cas Number:

95-49-8

Molecular formula:

C7H7Cl

IUPAC Name:

1-chloro-2-methylbenzene

Test animals

Species:

rat

Strain:

other: CrL : COBS CD (SD) BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Magate, Kent, UK
- Age at study initiation: mature females
- Weight at study initiation: 166 - 208 g
- Housing: in suspended polypropylene cages with stainless steel mesh floors and tops. Undercage plastic trays were lined with absorbent paper which was inspected and changed daily.
- Diet: Spratt's Laboratory Diet No.l, ad libitum
- Water: tap water , ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +- 4
- Humidity (%): 60 +- 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test substance was delivered at a controlled rate through a stainless steel metering valve (Nupro Company, 4800 East 345th Street, Willoughby, Ohio 44094, U.S.A.) to an all-glass concentric jet atomiser. The test substance was supplied from a central reservoir constructed from stainless steel which was pressurised to approximately 200 mm Hg using nitrogen from a compressed gas cylinder. Test substance was fed to 3 metering valves (one for each dose group) through stainless steel tubing (i.d. 5 mm). A flexible PVC tube was used to connect the metering valve to the glass atomiser.
Compressed air was delivered to the glass atomiser at 25 litres/min through a flowmeter with needle valve control, to generate an aerosol of 2-chlorotoluene. The atomiser was fitted into the top of a doublesurface glass condenser which was heated by steam generated from an electrically heated water boiler. The aerosol of 2-chlorotoluene was vaporised by contact with the heated glass surfaces of the condenser. The vapour passed into a glass flask before being delivered to the inlet duct of the exposure chamber. In order to minimise the risk of vapour condensing before reaching the chamber, diluent air was fed at 20 litres/min to a side arm of the atomiser and the air was heated as it passed through the condenser.

- Method of holding animals in test chamber: The rats were held during exposure in stainless steel mesh cages placed on supports in the chamber. The cages were subdivided to isolate each rat within an equal sized compartment such that 4 rats were placed in each cage. The rats were positioned on a different tier in the chambers daily.
-Room air was drawn through each chamber by an extract unit, air entering each chamber through a 50 mm i.d. inlet duct fitted with an in-line venturi nozzle used to measure the airflow. The extracted air was diluted and vented through a stack mounted on the roof of the building. The chambers were operated at an internal pressure of 2030 mm H^O below ambient to prevent the escape of test substance into the laboratory. The test vapour was introduced into the chamber inlet duct above the venturi nozzle.
- The target total airflow through each chamber was 250 litres/minute. At this flow rate the calculated 95% chamber equilibration time was 12 minutes.
The rats were held during exposure in stainless steel mesh cages placed on supports in the chamber. The cages were subdivided to isolate each rat within an equal sized compartment such that 4 rats were placed in each cage. The rats were positioned on a different tier in the chambers daily.

TEST ATMOSPHERE
- Brief description of analytical method used: Concentrations of 2-chlorotoluene in the test chambers were measured using a Miran 1 portable infra-red gas analyser fitted with a 20 m variable path-length gas cell (Wilks Scientific Corporation, 140 Water Street, Box 449, South Norwalk, Connecticut, U.S.A.).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The measured chamber concentrations of 2-chlorotoluene gave values close to the target concentration for all test groups on all days of exposure:
0, 1.1, 3,1; 9.0 mg/l

Details on mating procedure:

Animals were not mated during the study.

Duration of treatment / exposure:

days 6-19 of gestation

Frequency of treatment:

6 h/d

Duration of test:

days 6-19 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

9 mg/L air (nominal)

Doses / concentrations 2

Dose / conc.:

3 mg/L air (nominal)

Doses / concentrations 3

Dose / conc.:

1 mg/L air (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/L air (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent no treatment

Details on study design:

Sex: female
Duration of test: on day 20 of gestation the dams were killed

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on day 1, 3, 6, 10, 14, 17 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Not provided in the study report. However, based on findings at least kidney and lungs were examined

FOOD CONSUMPTION: Yes
- Food consumption was measured daily for each cage of animals

WATER CONSUMPTION: Yes
- Water consumption was measured daily for each cage of animals

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

No

Fetal examinations:

- External examinations: Yes:
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter

The following parameters were examined for foetuses:
- Number of distribution of live young
- Number of distribution of embryonic/foetal death
- individual foetal weight
- sex ratio

Statistics:

Statistical analyses were performed routinely on litter data and incidences of skeletal variants, using the litter as the basic sample unit and non-parametric methods (Jonckheere, Kruskal-Wallise) as these values rarely follow a normal distribution.

Indices:

Pregnancy rate was determined.

In assessing litter data pre-implantation loss was calculated for each litter as a percentage from the formula:

((Number of corpora lutea - Number of implantations)/Number of corpora lutea) x 100;

Post implantation loss was similarly calculated from the formula:
((Number of implantations - Number of live young)/Number of implantations) x 100


For all values expressed as a percentage or ratio, values were first calculated within the litter and the group values derived as a mean of individual litter percentages.
Calculation of separate mean A values (includes all surviving pregnant animals) and mean B values (includes all surviving dams with viable young), was not applicable in this study as there were no total litter losses.

Historical control data:

Not provided

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 9 mg/l all animals showed slight to moderate ataxia during exposure and occasional animals showed lachrymation and/or salivation.

At 3 mg/l all animals showed slight ataxia during exposure.

There were no signs at 1 mg/l that were observed specifically during the exposure periods.

General signs included brown fur staining among all animals at 9 mg/l and an increased incidence of animals showing some fur loss at 1 and 3 mg/l.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 9 mg/l mean bodyweight gain was markedly reduced.

Mean bodyweight gain at 3 mg/l was slightly reduced from the start of treatment through to Day 10 of pregnancy and parity with control animals was not subsequently regained.

Mean bodyweight gain at 1 mg/l was essentially similar to that of control animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 9 mg/l mean food consumption was significantly reduced from the start of treatment through to termination.

Mean values for food consumption at 3 mg/l were consistently slightly lower than control values although the differences were not statistically significant (P>0.05).

Mean food consumption 1 mg/l was essentially unaffected by treatment.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Mean water consumption was significantly increased at 3 and 9 mg/l from the start of treatment, with markedly increased values occurring at 9 mg/l.

At 1 mg/l mean water consumption was unaffected by treatment.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine 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):

Occasional macroscopic visceral changes observed at terminal autopsy appeared unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Slight intergroup differences among mean values for litter size and pre- and post implantation losses were neither statistically significant (P>0.05) nor dosage-related.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no instances of total resorption.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Dose descriptor:

NOAEC

Effect level:

1.1 mg/L air (analytical)

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 9 mg/l litter and mean foetal weight were significantly reduced (P<0.01 and P<0.001, respectively).
Litter and mean foetal weights at 1 and 3 mg/l appeared essentially unaffected by treatment. Marginally lower values for mean foetal weight relative to the control group probably reflected the slightly higher mean litter size, and hence greater intra-litter competition, that occurred in both groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

see above "Fetal body weight changes"

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At 9 mg/l there was an increased incidence of malformed foetuses mainly due to the occurrence of six foetuses, distributed among four litters, showing brachydactyly. One of these affected pups also showed brachymelia of one hind limb. The three other malformations at 9 mg/l involved single foetuses showing respectively microphthalmia, anophthalmia and cardiac ventricular septal defect. The brachydactyly malformation involved the loss of the claw and terminal phalanx of one or more digits. All six foetuses showed a single affected fore or hind paw. For five of the six foetuses the brachydactyly was associated with a terminal haemorrhagic area on the affected paw.

There were no malformed foetuses at 3 mg/l.

At 1 mg/l there were four malformed foetuses compared to three in the control group. The four malformed foetuses at 1 mg/l involved one showing brachygnathia, one showing retro-oesophageal aortic arch, one showing cardiac ventricular septal defect and one showing brachydactyly and brachymelia of all four limbs. The last mentioned malformation showed apparent similarity to that shown by six foetuses at 9 mg/l. Despite this as similar malformations have occurred spontaneously among control animals in previous studies and in the absence of any other evidence of adverse effects on young at 1 and 3 mg/l, it was considered that overall there was no conclusive indication of a treatment relationship.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The incidence of sternebral variants at 9 mg/l was significantly increased (P<0.001). This mainly reflected an increase in the incidence of foetuses with one or more unossified sternebra(e) and was probably associated with the reduced mean foetal weight observed in this group.

At 1 and 3 mg/l marginally higher incidences of sternebral variants in comparison to the control group was neither statistically significant (P>0.05) nor dosage-related.
At 9 mg/l the incidence of foetuses with extra thoraco-lumbar ribs was marginally higher than the control incidence; the difference was not statistically significant (P>0.05).
Incidences at 1 and 3 mg/l were unaffected.

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: yes, see above "external malformation".

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEC

Effect level:

3.1 mg/L air (analytical)

Basis for effect level:

other: teratogenicity

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

9 mg/L air (analytical)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Any other information on results incl. tables

Rats inhaled analyzed o-chlorotoluene concentrations of 0, 1, 3, or 9 mg/l for 6 hours/day from days 6 to 19 of pregnancy. Animals in the 3 mg/l group exhibited slight ataxia during exposure. Animals in the 9 mg/l group displayed ataxia, lacrimation and/or salivation as well as a brownish discoloration of the fur. Beginning at 3 mg/l, a dose-dependent reduction in feed intake and body weight gain was observed, as well as a dose-dependent increase in drinking water consumption. No treatment-related effects were seen for animals in the 1 mg/l group. The offspring of the 1 and 3 mg/l groups did not exhibit treatment-related effects. In the 9 mg/l group, a reduction in fetal and litter weights was observed. The incidence of fetal malformations was increased by 6 fetuses (distributed among 4 litters), which each exhibited brachdactylia on one of its fore or hind paws. 5 of these 6 fetuses simultaneously displayed terminal hemorrhaging in the affected paw. In correlation to the reduced mean fetal weight, delayed ossification led to an increased occurrence of skeletal variations. The incidence of visceral anomalies was unchanged, however.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, a NOAEL of 1.1. mg/L for maternal toxicity (based on reduced feed intake and body weight gain, and general toxicity in the mid and high dose group) and a NOAEL of 3.1 mg/l for developmental toxicity (teratogenicity) was established.

Executive summary:

On each of gestation days 6 to 19, 25 Charles River rats were transferred to exposure chambers and exposed to a an atmosphere of the test material at concentrations of 0, 1, 3, or 9 mg/l air for 6 hours (analytical concentrations were measured to be 0, 1.1, 1.3, 9.0 mg/l). All animals were killed on day 20 of gestation, dissected and examined for abnormalities and changes in maternal organs. The ovaries and uteri were examined to determine number of corpora lutea, number of resorptions, distribution of live young, number and ditribution of embryonic/foetal deaths, individual foetal weight and gross foetal abnormalities. Half of the pups in each litter were preserved for subsequent sectioning to discover visceral abnormalities; the remainder were fixed for skeletal examination.

Animals in the 3.1 mg/l group exhibited slight ataxia during exposure. Animals in the 9 mg/l group displayed ataxia, lacrimation and/or salivation as well as a brownish discoloration of the fur. Beginning at 3.1 mg/l, a dose-dependent reduction in feed intake and body weight gain was observed, as well as a dose-dependent increase in drinking water consumption. No treatment-related effects were seen for animals in the 1.1 mg/l group. The offspring of the 1.1 and 3.1 mg/l groups did not exhibit treatment-related effects. In the 9 mg/l group, a reduction in fetal and litter weights was observed. The incidence of fetal malformations was increased by 6 fetuses (distributed among 4 litters), which each exhibited brachydactyly on one of its fore or hind paws. Five of these 6 fetuses simultaneously displayed terminal hemorrhaging in the affected paw. Incorrelation to the reduced mean fetal weight, delayed ossification led to an increased occurence of skeletal variations. The incidence of visceral anomalies was unchanged.

The NOAEC for maternal toxicity is 1.1 mg/l, the NOAEC for teratogenicity is 3.1 mg/l.